Immunity-inducing agent

ABSTRACT

An immunity-inducing agent comprising as an effective ingredient(s) a polypeptide(s) selected from the polypeptides: (a) a polypeptide consisting essentially of not less than 7 consecutive amino acids in any one of the amino acid sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 44 in SEQUENCE LISTING; (b) a polypeptide having a sequence identity of not less than 90% with the polypeptide (a) and consisting essentially of not less than 7 amino acids; and (c) a polypeptide comprising the polypeptide (a) or (b) as a partial sequence thereof; which polypeptide(s) has/have an immunity-inducing activity/activities, or as an effective ingredient(s) a recombinant vector(s) which comprise(s) a polynucleotide(s) encoding the polypeptide(s) and is/are capable of expressing the polypeptide(s) in vivo, is useful as a therapeutic and/or prophylactic agent for cancer, and/or the like.

This application is a Divisional of U.S. patent application Ser. No.13/391,595 filed on Mar. 9, 2012, which is the national stage entry ofinternational application PCT/JP2010/064993 filed on Sep. 2, 2010, whichclaims priority to Application No. 2009-203489 filed in Japan on Sep. 3,2009, all of which are hereby expressly incorporated by reference intothe present application.

TECHNICAL FIELD

The present invention relates to a novel immunity-inducing agent usefulas a therapeutic and/or prophylactic agent for cancer.

BACKGROUND ART

Cancer is the commonest cause for death among all of the causes fordeath, and therapies carried out therefor at present are mainly surgicaltreatment, which may be carried out in combination with radiotherapyand/or chemotherapy. In spite of the developments of new surgicalmethods and discovery of new anti-cancer agents in recent years,treatment results of cancers have not been improved very much at presentexcept for some cancers. In recent years, by virtue of the developmentin molecular biology and cancer immunology, cancer antigens recognizedby cytotoxic T cells reactive with cancers, as well as the genesencoding the cancer antigens, were identified, and expectations forantigen-specific immunotherapies have been raised.

In immunotherapy, in order to reduce side effects, it is necessary thatthe peptide or protein to be recognized as the antigen exist hardly innormal cells and exist specifically in cancer cells. In 1991, Boon etal. of Ludwig Institute in Belgium isolated a human melanoma antigenMAGE 1, which is recognized by CD8-positive T cells, by acDNA-expression cloning method using an autologous cancer cell line andcancer-reactive T cells (Non-patent Document 1). Thereafter, the SEREX(serological identifications of antigens by recombinant expressioncloning) method, wherein tumor antigens recognized by antibodiesproduced in the living body of a cancer patient in response to thecancer of the patient himself are identified by application of a geneexpression cloning method, was reported (Patent Document 1, Non-patentDocument 2), and several cancer antigens have been isolated by thismethod. Using a part of the cancer antigens as targets, clinical testsfor cancer immunotherapy have started.

On the other hand, as in human, a number of tumors such as mammary glandtumor and squamous cell carcinoma are known in dogs and cats, and theyrank high also in the statistics of diseases in dogs and cats. However,at present, no therapeutic agent, prophylactic agent or diagnostic agentexists which is effective for cancers in dogs and cats. Most of tumorsin dogs and cats are realized by owners only after they advanced to growbigger, and in many cases, it is already too late to visit a hospital toreceive surgical excision of the tumor or administration of a human drug(an anticancer drug or the like), so that those dogs and cats often dieshortly after the treatment. Under such circumstances, if therapeuticagents and prophylactic agents for cancer effective for dogs and catsbecome available, their uses for canine cancers are expected to bedeveloped.

PDS5A (PDS5, regulator of cohesion maintenance, homolog A) is a proteinalso called SSC-112 which was identified as a cell cycle regulatorinvolved in distribution of chromosomes, and reported to show higherexpression in nasopharyngeal carcinoma, renal cancer, liver cancer and acertain type of breast cancer cells, compared to normal tissues (PatentDocument 2, Non-patent Documents 3 to 5). It has been reported that thegrowth of cancer cells can be suppressed by suppressing expression ofPDS5A in cancer cells using an antisense nucleic acid, ribozyme or siRNAagainst the PDS5A gene or using an antibody that specifically binds tothe PDS5A protein, and that cancer cells can be induced to causeapoptosis by administering the full-length PDS5A protein or a partialpeptide of the PDS5A protein (Patent Document 3). Further, in PatentDocument 3, increase in the mRNA level of the PDS5A protein in cancercells was confirmed. However, there is no report suggesting that thePDS5A protein and a partial peptide of the protein has an action toinduce immunity against cancer cells and hence the protein and a partialpeptide of the protein is useful for therapy or prophylaxis of cancer,and whether or not the PDS5A protein has a function as a marker that canbe used for diagnosis of cancer has not been confirmed.

PRIOR ART DOCUMENTS Patent Documents

-   [Patent Document 1] U.S. Pat. No. 5,698,396 B-   [Patent Document 2] WO2006/109943-   [Patent Document 3] WO2002/081641

Non-Patent Documents

-   [Non-patent Document 1] Science, 254: 1643-1647 (1991)-   [Non-patent Document 2] Proc. Natl. Acad. Sci. USA, 92: 11810-11813    (1995)-   [Non-patent Document 3] Gene. 17; 328: 187-96 (2004)-   [Non-patent Document 4] J. Cell. Sci. 15; 118 (Pt 10): 2133-41    (2005)-   [Non-patent Document 5] J. Cancer Res. Clin. Oncol.: 134(4):453-62    (2008)

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The present invention aims to discover a novel polypeptide useful for atherapeutic and/or prophylactic agent for cancer or useful for detectionof cancer, to provide the polypeptide for use in an immunity-inducingagent or in detection of cancer

Means for Solving the Problems

By the SEREX method using a canine breast cancer-derived cDNA libraryand serum obtained from a tumor-bearing dog, the present inventorsintensively studied to obtain a cDNA encoding a protein which binds toantibodies existing in the serum derived from a tumor-bearing livingbody, and, based on the cDNA, the canine PDS5 protein, a regulator ofcohesion maintenance, homolog A (hereinafter also referred to as PDS5A),having the amino acid sequence shown in SEQ ID NO:2 was prepared.Further, based on human and murine homologous genes of the obtainedgene, human PDS5A having the amino acid represented by SEQ ID NO:4 or 44(SEQ ID NO:4 corresponds to a partial sequence of SEQ ID NO:44) andmurine PDS5A having the amino acid sequence shown in SEQ ID NO:6 wereprepared. The present inventors then discovered that that these PDS5Aare specifically expressed in tissues or cells of breast cancer, braintumor, esophagus cancer, lung cancer, renal cancer, colon cancer,perianal adenocarcinoma, neuroblastoma and leukemia. Further, thepresent inventors discovered that, by administration of these PDS5A to aliving body, immunocytes against PDS5A can be induced in the livingbody, and a tumor in the living body expressing PDS5A can be regressed.Further, the present inventors discovered that a recombinant vectorwhich can express a polynucleotide encoding the full-length PDS5Aprotein or a fragment thereof can induce an anti-tumor effect againstcancer expressing PDS5A in the living body.

Further, the present inventors discovered that a partial peptide ofPDS5A has a capacity to be presented by antigen-presenting cells,thereby allowing activation and growth of cytotoxic T cells specific tothe peptide (immunity-inducing activity), and therefore that the peptideis useful for therapy and/or prophylaxis of cancer, and, further, thatantigen-presenting cells which have contacted with the peptide and Tcells which have contacted with the antigen-presenting cells are usefulfor therapy and/or prophylaxis of cancer, thereby completing the presentinvention.

Thus, the present invention has the following characteristics.

(1) An immunity-inducing agent comprising as an effective ingredient(s)at least one polypeptide selected from the polypeptides (a) to (c)below, the polypeptide(s) having an immunity-inducingactivity/activities, or as an effective ingredient(s) a recombinantvector(s) which comprise(s) a polynucleotide(s) encoding thepolypeptide(s) and is/are capable of expressing the polypeptide(s) invivo:

(a) a polypeptide consisting essentially of not less than 7 consecutiveamino acids in any one of the amino acid sequences shown in SEQ IDNOs:2, 4, 6, 8, 10, 12 and 44 in SEQUENCE LISTING;

(b) a polypeptide having a sequence identity of not less than 90% withthe polypeptide (a) and consisting essentially of not less than 7 aminoacids; and

(c) a polypeptide comprising the polypeptide (a) or (b) as a partialsequence thereof.

(2) The immunity-inducing agent according to (1), wherein thepolypeptide (b) has a sequence identity of not less than 95% with thepolypeptide (a).(3) The immunity-inducing agent according to (1), wherein each of thepolypeptide(s) having an immunity-inducing activity/activities is apolypeptide consisting essentially of not less than 7 consecutive aminoacids in any one of the amino acid sequences shown in SEQ ID NOs:2, 4,6, 8, 10, 12 and 44, or a polypeptide comprising the polypeptide as apartial sequence thereof; or a polypeptide having the same amino acidsequence as a polypeptide consisting essentially of not less than 7consecutive amino acids in any one of the amino acid sequences shown inSEQ ID NOs:2, 4, 6, 8, 10, 12 and 44 except that one or several aminoacids are deleted, substituted and/or added, or a polypeptide comprisingthe polypeptide as a partial sequence thereof.(4) The immunity-inducing agent according to (3), wherein each of thepolypeptide(s) having an immunity-inducing activity/activities is apolypeptide having any one of the amino acid sequences shown in SEQ IDNOs:2, 4, 6, 8, 10, 12 and 44 in SEQUENCE LISTING.(5) The immunity-inducing agent according to (3), wherein each of thepolypeptide(s) having an immunity-inducing activity/activities is apolypeptide consisting essentially of not less than 7 consecutive aminoacids in the region of aa111-140, aa211-240, aa248-278, aa327-357,aa459-522, aa909-972, aa959-1022, aa994-1057 or aa1018-1080 in any oneof the amino acid sequences shown in SEQ ID NOs:2, 6, 8, 10, 12 and 44in SEQUENCE LISTING, or a polypeptide comprising the polypeptide as apartial sequence thereof; or a polypeptide having the same amino acidsequence as a polypeptide consisting essentially of not less than 7consecutive amino acids in the region of aa111-140, aa211-240,aa248-278, aa327-357, aa459-522, aa909-972, aa959-1022, aa994-1057 oraa1018-1080 in any one of the amino acid sequences shown in SEQ IDNOs:2, 6, 8, 10, 12 and 44 in SEQUENCE LISTING except that one orseveral amino acids are deleted, substituted and/or added, or apolypeptide comprising the polypeptide as a partial sequence thereof.(6) The immunity-inducing agent according to (5), wherein each of thepolypeptide(s) having an immunity-inducing activity/activities is apolypeptide having any one of the amino acid sequences shown in SEQ IDNOs:27 to 35 in SEQUENCE LISTING, or a polypeptide comprising thepolypeptide as a partial sequence thereof and having 10 to 12 amino acidresidues; or a polypeptide having the same amino acid sequence as apolypeptide having any one of the amino acid sequences shown in SEQ IDNOs:27 to 35 in SEQUENCE LISTING except that one or several amino acidsare deleted, substituted and/or added, or a polypeptide comprising thepolypeptide as a partial sequence thereof and having 10 to 12 amino acidresidues.(7) The immunity-inducing agent according to any one of (1) to (6), forprophylaxis of a cancer in an animal.(8) The immunity-inducing agent according to (5) or (6), for therapy ofa cancer in an animal.(9) The immunity-inducing agent according to (7) or (8), wherein thecancer is a cancer expressing PDS5A.(10) The immunity-inducing agent according to any one of (7) to (9),wherein the cancer is breast cancer, brain tumor, esophagus cancer, lungcancer, renal cancer, colon cancer, perianal adenocarcinoma,neuroblastoma or leukemia.(11) The immunity-inducing agent according to any one of (1) to (10),further comprising an immunoenhancer.(12) An isolated antigen-presenting cell comprising a complex betweenthe polypeptide having an immunity-inducing activity and an MHCmolecule.(13) An isolated T cell which selectively binds to a complex between thepolypeptide having an immunity-inducing activity and an MHC molecule.(14) A polypeptide having any one of the amino acid sequences shown inSEQ ID NOs:27 to 35 in SEQUENCE LISTING, or a polypeptide comprising thepolypeptide as a partial sequence thereof and having 10 to 12 amino acidresidues; or a polypeptide having the same amino acid sequence as apolypeptide having any one of the amino acid sequences shown in SEQ IDNOs:27 to 35 in SEQUENCE LISTING except that one or several amino acidsare deleted, substituted and/or added, or a polypeptide comprising thepolypeptide as a partial sequence thereof and having 10 to 12 amino acidresidues, which polypeptide has an immunity-inducing activity.(15) A method for detecting a cancer, the method comprising measurementof expression of a polypeptide having any one of the amino acidsequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 44 in SEQUENCELISTING or a polypeptide having a sequence identity of not less than 90%with the polypeptide, in a sample separated from a living body.(16) A method for inducing immunity, the method comprising administeringto an individual at least one polypeptide selected from the polypeptides(a) to (c) below, the polypeptide(s) having an immunity-inducingactivity/activities, or a recombinant vector(s) which comprise(s) apolynucleotide(s) encoding the polypeptide(s) and is/are capable ofexpressing the polypeptide(s) in vivo:

(a) a polypeptide consisting essentially of not less than 7 consecutiveamino acids in any one of the amino acid sequences shown in SEQ IDNOs:2, 4, 6, 8, 10, 12 and 44 in SEQUENCE LISTING;

(b) a polypeptide having a sequence identity of not less than 90% withthe polypeptide (a) and consisting essentially of not less than 7 aminoacids; and

(c) a polypeptide comprising the polypeptide (a) or (b) as a partialsequence thereof.

Effect of the Invention

By the present invention, a novel immunity-inducing agent useful fortherapy and/or prophylaxis and/or the like of cancer is provided. Asparticularly described in later-mentioned Examples, by administering thepolypeptide used in the present invention to a living body, immunocytescan be induced in the living body, and a cancer which has alreadyoccurred can be reduced or regressed. Therefore, the polypeptide isuseful for therapy and/or prophylaxis of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression patterns of the identified PDS5A gene incanine normal tissues, tumor tissues and tumor cell lines. Referencenumeral 1, the expression patterns of the canine PDS5A gene in variouscanine tissues and cell lines; reference numeral 2, the expressionpatterns of the canine GAPDH gene in various canine tissues and celllines.

FIG. 2 shows the expression patterns of the identified PDS5A gene inhuman normal tissues, tumor tissues and tumor cell lines. Referencenumeral 3, the expression patterns of the human PDS5A gene in varioushuman tissues and cell lines; reference numeral 4, the expressionpatterns of the human GAPDH gene in various human tissues and celllines.

FIG. 3 shows the expression patterns of the identified PDS5A gene inmurine normal tissues, tumor tissues and tumor cell lines. Referencenumeral 5, the expression patterns of the murine PDS5A gene in variousmurine tissues and cell lines; reference numeral 6, the expressionpatterns of the murine GAPDH gene in various murine tissues and celllines.

FIG. 4 is a graph showing that an anti-tumor effect (therapeutic model:neuroblastoma cell line) was observed by administration of PDS5A.Immunization was carried out with a vector alone or a plasmid encodingPDS5A using a gene gun, and the evaluation was carried out based on thearea of the cancerous part and the ratio of living mice. For each group,10 individuals of mice were used. The mice were observed twice a week.The data are represented by the mean value ±SD. Reference numeral 7, thegroup wherein a plasmid vector was administered; reference numeral 8,the group wherein a plasmid encoding PDS5A was administered.

FIG. 5 is a graph showing that an anti-tumor effect (prophylactic model:neuroblastoma cell line) was observed by administration of PDS5A.Immunization was carried out with a vector alone or a plasmid encodingPDS5A using a gene gun, and the evaluation was carried out based on thearea of the cancerous part and the ratio of living mice. For each group,10 individuals of mice were used. The mice were observed twice a week.The data are represented by the mean value ±SD. Reference numeral 9, thegroup wherein a plasmid vector was administered; reference numeral 10,the group wherein a plasmid encoding PDS5A was administered.

FIG. 6 shows the ratio of living mice in the experiment in FIG. 4.Reference numeral 11, the group wherein a plasmid vector wasadministered; reference numeral 12, the group wherein a plasmid encodingPDS5A was administered.

FIG. 7 shows the ratio of living mice in the experiment in FIG. 5.Reference numeral 13, the group wherein a plasmid vector wasadministered; reference numeral 14, the group wherein a plasmid encodingPDS5A was administered.

FIG. 8 is a graph showing that an anti-tumor effect (therapeutic model:colon cancer cell line) was observed by administration of PDS5A.Immunization was carried out with a vector alone or a plasmid encodingPDS5A using a gene gun, and the evaluation was carried out based on thearea of the cancerous part and the ratio of living mice. For each group,10 individuals of mice were used. The mice were observed twice a week.The data are represented by the mean value ±SD. Reference numeral 15,the group wherein a plasmid vector was administered; reference numeral16, the group wherein a plasmid encoding PDS5A was administered.

FIG. 9 is a graph showing that an anti-tumor effect (prophylactic model:colon cancer cell line) was observed by administration of PDS5A.Immunization was carried out with a vector alone or a plasmid encodingPDS5A using a gene gun, and the evaluation was carried out based on thearea of the cancerous part and the ratio of living mice. For each group,10 individuals of mice were used. The mice were observed twice a week.The data are represented by the mean value ±SD. Reference numeral 17,the group wherein a plasmid vector was administered; reference numeral18, the group wherein a plasmid encoding PDS5A was administered.

FIG. 10 shows the ratio of living mice in the experiment in FIG. 8.Reference numeral 19, the group to which a plasmid vector wasadministered; reference numeral 20, the group to which a plasmidencoding PDS5A was administered.

FIG. 11 shows the ratio of living mice in the experiment in FIG. 9.Reference numeral 21, the group to which a plasmid vector wasadministered; reference numeral 22, the group to which a plasmidencoding PDS5A was administered.

FIG. 12 is a diagram showing that CD8-positive T cells specific to eachof the polypeptides having the amino acid sequences shown in SEQ IDNOs:27 to 35 in SEQUENCE LISTING recognize the complex between thepolypeptide and HLA-A0201, and produce IFN-γ. In FIG. 12, the referencenumerals 25 to 33 along the abscissa indicate the abilities ofHLA-A0201-positive CD8-positive T cells to produce IFN-γ in response tostimulation by T2 cells pulsed with the respective peptides of SEQ IDNOs:27 to 35. The reference numeral 23 shows a result obtained when theabove treatment was carried out without addition of a polypeptide, andthe reference numeral 24 shows a result obtained when the abovetreatment was carried out with addition of the polypeptide shown in SEQID NO:36, which is outside the scope of the present invention.

FIG. 13 is a diagram showing the cytotoxic activities, against cancercells, of CD8-positive T cells specific to each of the polypeptideshaving the amino acid sequences shown in SEQ ID NOs:27 to 35 in SEQUENCELISTING. In FIG. 13, the reference numerals 36 to 44 along the abscissaindicate the cytotoxic activities, against T98G cells, ofHLA-A0201-positive CD8-positive T cells stimulated with the respectivepeptides of SEQ ID NOs:27 to 35. The reference numeral 34 shows thecytotoxic activity of CD8-positive T cells induced without addition of apolypeptide, and the reference numeral 35 shows the cytotoxic activityof CD8-positive T cells induced using a negative control peptide (SEQ IDNO:36).

BEST MODE FOR CARRYING OUT THE INVENTION

Examples of the polypeptide contained in the immunity-inducing agent ofthe present invention as an effective ingredient include the followings.In the present invention, the term “polypeptide” means a molecule formedby a plurality of amino acids linked together by peptide bonds, andincludes not only polypeptide molecules having large numbers of aminoacids constituting them, but also low-molecular-weight molecules havingsmall numbers of amino acids (oligopeptides), and full-length proteins.In the present invention, the full-length PDS5A proteins having theamino acid sequences shown in SEQ ID NO:2, 4, 6, 8, 10, 12 and 44 arealso included therein.

(a) A polypeptide which consists essentially of not less than 7consecutive amino acids in a polypeptide having the amino acid sequenceshown in SEQ ID NO:2, 4, 6, 8, 10, 12 or 44 in SEQUENCE LISTING, and hasan immunity-inducing activity.

(b) a polypeptide which has a sequence identity of not less than 90%with the polypeptide (a), consists essentially of not less than 7 aminoacids, and has an immunity-inducing activity.

(c) a polypeptide which comprises the polypeptide (a) or (b) as apartial sequence thereof, and has an immunity-inducing activity.

In the present invention, the term “having an amino acid sequence” meansthat amino acid residues are arrayed in such an order. Therefore, forexample, “polypeptide having the amino acid sequence shown in SEQ IDNO:2” means the polypeptide having the amino acid sequence of Met AspPhe Thr . . . (snip) . . . Asp Leu Gln Arg shown in SEQ ID NO:2, whichpolypeptide has a size of 1337 amino acid residues. Further, forexample, “polypeptide having the amino acid sequence shown in SEQ IDNO:2” may be abbreviated as “polypeptide of SEQ ID NO:2”. This alsoapplies to the term “having a base sequence”. In this case, the term“having” may be replaced with the expression “essentially consistingof”.

As used herein, the term “immunity-inducing activity” means an abilityto induce immunocytes which secrete cytokines such as interferon in aliving body.

Whether or not the polypeptide has an immunity-inducing activity can beconfirmed using, for example, the known ELISPOT assay. Moreparticularly, for example, as described in the Examples below, cellssuch as peripheral blood mononuclear cells are obtained from a livingbody to which a polypeptide whose immunity-inducing activity is to beevaluated was administered, which cells are then cocultured with thepolypeptide, followed by measuring the amount(s) of a cytokine(s)produced by the cells using a specific antibody/antibodies, therebymeasuring the number of immunocytes in the cells, which enablesevaluation of the immunity-inducing activity.

Alternatively, as described in the later-mentioned Examples, when arecombinant polypeptide in any of (a) to (c) described above isadministered to a tumor-bearing animal, the tumor can be regressed byits immunity-inducing activity. Thus, the above immunity-inducingactivity can be evaluated also as an ability to suppress the growth ofcancer cells or to cause reduction or disappearance of a cancer tissue(tumor) (hereinafter referred to as “anti-tumor activity”). Theanti-tumor activity of a polypeptide can be confirmed by, for example,as more particularly described in the Examples below, observation ofwhether or not a tumor is reduced when the polypeptide was actuallyadministered to a tumor-bearing living body.

Alternatively, the anti-tumor activity of a polypeptide can be evaluatedalso by observation of whether or not T cells stimulated with thepolypeptide (that is, T cells brought into contact withantigen-presenting cells presenting the polypeptide) show a cytotoxicactivity against tumor cells in vitro. The contact between the T cellsand the antigen-presenting cells can be carried out by coculture of theboth in a liquid medium, as mentioned below. Measurement of thecytotoxic activity can be carried out by, for example, the known methodcalled ⁵¹Cr release assay described in Int. J. Cancer, 58: p 317, 1994.In cases where the polypeptide is to be used for therapy and/orprophylaxis of cancer, the evaluation of the immunity-inducing activityis preferably carried out using the anti-tumor activity as an index,although the index is not restricted.

The amino acid sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 44in SEQUENCE LISTING are the amino acid sequences of the PDS5A proteinswhich were isolated, by the SEREX method using a canine testis-derivedcDNA library and serum of a tumor-bearing dog, as a polypeptide thatspecifically binds to an antibody existing in the serum of thetumor-bearing dog and homologous factors of the polypeptide in human(SEQ ID NOs:4 and 44), mouse (SEQ ID NO:6), cow (SEQ ID NO:8), horse(SEQ ID NO:10) and chicken (SEQ ID NO:12) (see Example 1). Human PDS5A,which is a human homologous factor of canine PDS5A, has a sequenceidentity of 94% in terms of the base sequence and 99% in terms of theamino acid sequence; murine PDS5A, which is a murine homologous factor,has a sequence identity of 91% in terms of the base sequence and 99% interms of the amino acid sequence; bovine PDS5A, which is a bovinehomologous factor, has a sequence identity of 95% in terms of the basesequence and 99% in terms of the amino acid sequence; equine PDS5A,which is an equine homologous factor, has a sequence identity of 96% interms of the base sequence and 99% in terms of the amino acid sequence;and chicken PDS5A, which is a chicken homologous factor, has a sequenceidentity of 83% in terms of the base sequence and 98% in terms of theamino acid sequence.

The polypeptide (a) is a polypeptide which consists essentially of notless than 7 consecutive, preferably 8, 9 or not less than 10 consecutiveamino acids in the polypeptide having the amino acid sequence shown inSEQ ID NO:2, 4, 6, 8, 10, 12 or 44, and has an immunity-inducingactivity. The polypeptide especially preferably has the amino acidsequence shown in SEQ ID NO:2, 4, 6, 8, 10, 12 or 44. As is known in theart, a polypeptide having not less than about 7 amino acid residues canexert its antigenicity and immunogenicity. Thus, a polypeptide havingnot less than 7 consecutive amino acid residues in the amino acidsequence shown in SEQ ID NO:2, 4, 6, 8, 10, 12 or 44 can have animmunity-inducing activity, so that it can be used for preparation ofthe immunity-inducing agent of the present invention.

As a principle of immune induction by administration of a cancerantigenic polypeptide, the following process is known: a polypeptide isincorporated into an antigen-presenting cell and then degraded intosmaller fragments by peptidases in the cell, followed by presentation ofthe fragments on the surface of the cell. The fragments are thenrecognized by a cytotoxic T cell or the like, which selectively killscells presenting the antigen. The size of the polypeptide presented onthe surface of the antigen-presenting cell is relatively small and about7 to 30 amino acids. Therefore, from the viewpoint of presenting thereofon the surface of the antigen-presenting cell, one preferred mode of theabove-described polypeptide (a) is a polypeptide composed of about 7 to30 consecutive amino acids in the amino acid sequence shown in SEQ IDNO:2, 4, 6, 8, 10, 12 or 44, and more preferably, a polypeptide composedof about 8 to 30 or about 9 to 30 amino acids is sufficient as thepolypeptide (a). In some cases, these relatively small polypeptides arepresented directly on the surface of the antigen-presenting cell withoutbeing incorporated into the antigen-presenting cells.

Further, since a polypeptide incorporated into an antigen-presentingcell is cleaved at random sites by peptidases in the cell to yieldvarious polypeptide fragments, which are then presented on the surfaceof the antigen-presenting cell, administration of a large polypeptidesuch as the full-length region of SEQ ID NO:2, 4, 6, 8, 10, 12 or 44inevitably causes production of polypeptide fragments by degradationthereof in the antigen-presenting cell, which fragments are effectivefor immune induction via the antigen-presenting cell. Therefore, alsofor immune induction via antigen-presenting cells, a large polypeptidecan be preferably used, and the polypeptide may be composed of not lessthan 30, preferably not less than 100, more preferably not less than200, still more preferably not less than 250 amino acids. Thepolypeptide may be still more preferably composed of the full-lengthregion of SEQ ID NO:2, 4, 6, 8, 10, 12 or 44.

Further, the polypeptides of the present invention can be checked with achecking medium by which epitope peptides having binding motifs ofvarious types of HLA and consisting essentially of 8 to 12, preferably 9to 10 amino acids can be searched, for example, HLA Peptide BindingPredictions (http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html) inBioinformatics & Molecular Analysis Selection (BIMAS), to screenpeptides which may be epitope peptides. More particularly, a polypeptideconsisting essentially of not less than 7 consecutive amino acids in theregion of amino acid residue positions aa111-140, aa211-240, aa248-278,aa327-357, aa459-522, aa909-972, aa959-1022, aa994-1057 or aa1018-1080in the amino acid sequence shown in SEQ ID NO:2, 6, 8, 10, 12 or 44 ispreferred, and, in the polypeptide of SEQ ID NO:4 or 44, the polypeptideshown in any of SEQ ID NOs:27 to 35, or a polypeptide which comprises apolypeptide having the amino acid sequence shown in any of SEQ ID NOs:27to 35 as a partial sequence and has 10 to 12 amino acid residues is morepreferred.

The polypeptide (b) is the same polypeptide as the polypeptide (a)except that a small number of (preferably, one or several) amino acidresidues are substituted, deleted and/or inserted, which has a sequenceidentity of not less than 90%, preferably not less than 95%, morepreferably not less than 98%, still more preferably not less than 99% ornot less than 99.5% to the original sequence and has animmunity-inducing activity. It is well known in the art that, ingeneral, there are cases where a protein antigen retains almost the sameantigenicity as the original protein even if the amino acid sequence ofthe protein is modified such that a small number of amino acids aresubstituted, deleted and/or inserted. Therefore, since the polypeptide(b) may also exert an immunity-inducing activity, it can be used forpreparation of the immunity-inducing agent of the present invention.Further, the polypeptide (b) is also preferably the same polypeptide asone having the amino acid sequence shown in SEQ ID NO:2, 4, 6, 8, 10, 12or 44 except that one or several amino acid residues are substituted,deleted and/or inserted. As used herein, the term “several” means aninteger of 2 to 10, preferably an integer of 2 to 6, more preferably aninteger of 2 to 4.

As used herein, the term “sequence identity” of amino acid sequences orbase sequences means the value calculated by aligning two amino acidsequences (or base sequences) to be compared such that the number ofmatched amino acid residues (or bases) is maximum between the amino acidsequences (or base sequences), and dividing the number of matched aminoacid residues (or the number of matched bases) by the total number ofamino acid residues (or the total number of bases), which value isrepresented as a percentage. When the alignment is carried out, a gap(s)is/are inserted into one or both of the two sequences to be compared asrequired. Such alignment of sequences can be carried out using awell-known program such as BLAST, FASTA or CLUSTAL W. When a gap(s)is/are inserted, the above-described total number of amino acid residuesis the number of residues calculated by counting one gap as one aminoacid residue. When the thus counted total number of amino acid residuesis different between the two sequences to be compared, the sequenceidentity (%) is calculated by dividing the number of matched amino acidresidues by the total number of amino acid residues in the longersequence.

The 20 types of amino acids constituting naturally occurring proteinsmay be classified into groups in each of which similar properties areshared, for example, into neutral amino acids with side chains havinglow polarity (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acidshaving hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidicamino acids (Asp, Glu), basic amino acids (Arg, Lys, His) and aromaticamino acids (Phe, Tyr, Trp). It is known that, in most cases,substitutions of amino acids within the same group do not change theproperties of the polypeptide. Therefore, in cases where an amino acidresidue(s) in the polypeptide (a) of the present invention is/aresubstituted, the probability that the immunity-inducing activity can bemaintained may be increased by introducing the substitution(s) withinthe same group, which is preferred.

As the polypeptide (b), which corresponds to the above-described epitopepeptide, a polypeptide which is the same as the polypeptide consistingessentially of not less than 7 consecutive amino acids in the region ofaa111-140, aa211-240, aa248-278, aa327-357, aa459-522, aa909-972,aa959-1022, aa994-1057 or aa1018-1080 in any one of the amino acidsequences shown in SEQ ID NOs:2, 6, 8, 10, 12 and 44 except that one orseveral amino acids are deleted, substituted and/or added, or apolypeptide comprising the polypeptide as a partial sequence thereof andhaving an immunity-inducing activity is preferred, and, in thepolypeptide of SEQ ID NO:4 or 44, a polypeptide which is the same as thepolypeptide having the amino acid sequence shown in any of SEQ ID NOs:27to 35 except that one or several amino acids are deleted, substitutedand/or added, or a polypeptide comprising the polypeptide as a partialsequence and having 10 to 12 amino acid residues is more preferred.

The polypeptide (c) comprises the polypeptide (a) or (b) as a partialsequence and has an immunity-inducing activity. That is, the polypeptide(c) has another/other amino acid(s) or polypeptide(s) added at one orboth ends of the polypeptide (a) or (b), and has an immunity-inducingactivity. Such a polypeptide can also be used for preparation of theimmunity-inducing agent of the present invention.

As the polypeptide (c), which corresponds to the above-describedepitope, a polypeptide comprising as a partial sequence the polypeptideconsisting essentially of not less than 7 consecutive amino acids in theregion of aa111-140, aa211-240, aa248-278, aa327-357, aa459-522,aa909-972, aa959-1022, aa994-1057 or aa1018-1080 in any one of the aminoacid sequences shown in SEQ ID NOs:2, 6, 8, 10, 12 and 44 is preferred,and, in the polypeptide of SEQ ID NO:4 or 44, a polypeptide comprisingas a partial sequence: a polypeptide which is the same as thepolypeptide having the amino acid sequence shown in any of SEQ ID NOs:27to 35 except that one or several amino acids are deleted, substitutedand/or added; or a polypeptide comprising the polypeptide as a partialsequence and having 10 to 12 amino acid residues; is more preferred.

The above-described polypeptides can be synthesized by, for example, achemical synthesis method such as the Fmoc method(fluorenylmethyloxycarbonyl method) or the tBoc method(t-butyloxycarbonyl method). Further, they can be synthesized byconventional methods using various types of commercially availablepeptide synthesizers. Further, the polypeptide of interest can beobtained using known genetic engineering techniques, by preparing apolynucleotide encoding the above polypeptide and incorporating thepolynucleotide into an expression vector, which is then introduced intoa host cell, followed by allowing the polypeptide to be produced in thehost cell.

The polynucleotide encoding the above polypeptide can be easily preparedby a known genetic engineering technique or a conventional method usinga commercially available nucleic acid synthesizer. For example, DNAhaving the base sequence shown in SEQ ID NO:1 can be prepared bycarrying out PCR using a canine chromosomal DNA or cDNA library as atemplate, and a pair of primers designed such that the base sequenceshown in SEQ ID NO:1 can be amplified therewith. DNA having the basesequence of SEQ ID NO:3 or 43 can be similarly prepared by using a humanchromosomal DNA or cDNA library as the template. The reaction conditionsfor the PCR can be set appropriately, and examples thereof include, butare not limited to, repeating the reaction process of 94° C. for 30seconds (denaturation), 55° C. for 30 seconds to 1 minute (annealing)and 72° C. for 2 minutes (extension) for, for example, 30 cycles,followed by the reaction at 72° C. for 7 minutes. Further, the desiredDNA can be isolated by preparing an appropriate probe(s) or primer(s)based on the information of the base sequences and the amino acidsequences shown in SEQ ID NO:1, 3, 5, 7, 9, 11 and 43 in SEQUENCELISTING in the present specification, and screening a cDNA library ofdog, human or the like using the probe(s) or primer(s). The cDNA libraryis preferably prepared from a cell, organ or tissue expressing theprotein of SEQ ID NO:2, 4, 6, 8, 10, 12 or 44. The above-describedoperations such as preparation of a probe(s) or primer(s), constructionof a cDNA library, screening of a cDNA library and cloning of a gene ofinterest are known to those skilled in the art, and can be carried outaccording to the methods described in Molecular Cloning, Second Edition;Current Protocols in Molecular Biology; and/or the like. From the thusobtained DNA, DNA encoding the polypeptide (a) can be obtained. Further,since the codons encoding each amino acid are known, the base sequenceof a polynucleotide encoding a specific amino acid sequence can beeasily specified. Therefore, since the base sequence of a polynucleotideencoding the polypeptide (b) or polypeptide (c) can also be easilyspecified, such a polynucleotide can also be easily synthesized using acommercially available nucleic acid synthesizer according to aconventional method.

The host cells are not restricted as long as those can express theabove-described polypeptide, and examples thereof include, but are notlimited to, prokaryotic cells such as E. coli; and eukaryotic cells suchas mammalian cultured cells including monkey kidney cells COS I_(—) andChinese hamster ovary cells CHO; budding yeast; fission yeast; silkwormcells; and Xenopus laevis egg cells.

In cases where prokaryotic cells are used as the host cells, anexpression vector in which an origin that enables replication of thevector in a prokaryotic cell, promoter, ribosome binding site, DNAcloning site, terminator and/or the like is/are contained is used.Examples of the expression vector for E. coli include the pUC system,pBluescriptII, pET expression system and pGEX expression system. Byincorporating a DNA encoding the above polypeptide into such anexpression vector and transforming prokaryotic host cells with thevector, followed by culturing the resulting transformants, thepolypeptide encoded by the DNA can be expressed in the prokaryotic hostcells. In this process, the polypeptide can also be expressed as afusion protein with another protein.

In cases where eukaryotic cells are used as the host cells, anexpression vector for eukaryotic cells having a promoter, splicing site,poly(A) addition site and/or the like is used as the expression vector.Examples of such an expression vector include pKA1, pCDM8, pSVK3, pMSG,pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, pMSG and pYES2. In thesame manner as described above, by incorporating a DNA encoding theabove polypeptide into such an expression vector and transformingeukaryotic host cells with the vector, followed by culturing theresulting transformants, the polypeptide encoded by the DNA can beexpressed in the eukaryotic host cells. In cases where pIND/V5-His,pFLAG-CMV-2, pEGFP-N1, pEGFP-C1 or the like is used as the expressionvector, the above polypeptide can be expressed as a fusion proteinwherein a tag such as a His tag, FLAG tag, myc tag, HA tag or GFP wasadded.

For the introduction of the expression vector into the host cells, awell-known method such as electroporation, the calcium phosphate method,the liposome method or the DEAE dextran method may be used.

Isolation and purification of the polypeptide of interest from the hostcells can be carried out by a combination of known separationoperations. Examples of the known separation operations include, but arenot limited to, treatment with a denaturant such as urea or with asurfactant; ultrasonication treatment; enzyme digestion; salting-out orsolvent fractional precipitation; dialysis; centrifugation;ultrafiltration; gel filtration; SDS-PAGE; isoelectric focusing;ion-exchange chromatography; hydrophobic chromatography; affinitychromatography; and reversed-phase chromatography.

The polypeptides obtained by the above method also include, as mentionedabove, those in the form of a fusion protein with another arbitraryprotein. Examples thereof include fusion proteins with glutathionS-transferase (GST) and with a His tag. Such a polypeptide in the formof a fusion protein is also included within the scope of the presentinvention as the above-described polypeptide (c). Further, in somecases, a polypeptide expressed in a transformed cell is modified invarious ways in the cell after translation. Such a post-translationallymodified polypeptide is also included within the scope of the presentinvention as long as it has an immunity-inducing activity. Examples ofsuch a post-translational modification include: elimination ofN-terminal methionine; N-terminal acetylation; glycosylation; limiteddegradation by an intracellular protease; myristoylation;isoprenylation; and phosphorylation.

As described more particularly in the later-mentioned Examples, byadministration of the polypeptide having an immunity-inducing activityor an expression vector comprising the gene encoding the polypeptide toa tumor-bearing living body, an already existing tumor can be regressed.Further, by administration of the polypeptide having animmunity-inducing activity or the gene encoding the polypeptide to aliving body before occurrence of cancer, development of a tumor can beprevented. Therefore, the immunity-inducing agent of the presentinvention can be used as a therapeutic and/or prophylactic agent forcancer. Further, the polypeptide having an immunity-inducing activitycan be used for a method of therapy and/or prophylaxis of cancer byimmune induction.

As used herein, the terms “tumor” and “cancer” mean a malignantneoplasm, and are used interchangeably

In this case, the cancer to be treated is not restricted as long asPDS5A is expressed in the cancer, and the cancer is preferably breastcancer, brain tumor, esophagus cancer, lung cancer, renal cancer, coloncancer, perianal adenocarcinoma, neuroblastoma or leukemia.

The subject animal is preferably a mammal, more preferably a mammal suchas a primate, pet animal, domestic animal or sport animal, especiallypreferably human, dog or cat.

The administration route of the immunity-inducing agent of the presentinvention to a living body may be either oral administration orparenteral administration, and is preferably parenteral administrationsuch as intramuscular administration, subcutaneous administration,intravenous administration or intraarterial administration. In caseswhere the immunity-inducing agent is used for therapy of cancer, it maybe administered to a regional lymph node in the vicinity of the tumor tobe treated, as described in the Examples below, in order to enhance itsanticancer activity. The dose may be any dose as long as the dose iseffective for immune induction, and, for example, in cases where theagent is used for therapy and/or prophylaxis of cancer, the dose may beone effective for therapy and/or prophylaxis of the cancer. The doseeffective for therapy and/or prophylaxis of cancer is appropriatelyselected depending on the size and symptoms of the tumor and the like,and the effective dose is usually, 0.0001 μg to 1000 μg, preferably0.001 μg to 1000 μg per subject animal per day, which may beadministered once or in several times. The agent is preferablyadministered in several times, every several days to several months. Asconcretely shown in the Examples below, the immunity-inducing agent ofthe present invention can cause regression of an already occurred tumor.Therefore, since the agent can exert its anticancer activity alsoagainst a small number of cancer cells at an early stage, development orrecurrence of cancer can be prevented by using the agent beforedevelopment of the cancer or after therapy for the cancer. That is, theimmunity-inducing agent of the present invention is effective for boththerapy and prophylaxis of cancer.

The immunity-inducing agent of the present invention may contain only apolypeptide or may be formulated by being mixed as appropriate with anadditive such as a pharmaceutically acceptable carrier, diluent orvehicle suitable for each administration mode. Formulation methods andadditives which may be used are well-known in the field of formulationof pharmaceuticals, and any of the methods and additives may be used.Specific examples of the additives include, but are not limited to,diluents such as physiological buffer solutions; vehicles such as sugar,lactose, corn starch, calcium phosphate, sorbitol and glycine; binderssuch as syrup, gelatin, gum arabic, sorbitol, polyvinyl chloride andtragacanth; and lubricants such as magnesium stearate, polyethyleneglycol, talc and silica. Examples of the formulation include oralpreparations such as tablets, capsules, granules, powders and syrups;and parenteral preparations such as inhalants, injection solutions,suppositories and solutions. These formulations may be prepared bycommonly known production methods.

The immunity-inducing agent of the present invention may be used incombination with an immunoenhancer capable of enhancing the immuneresponse in a living body. The immunoenhancer may be contained in theimmunity-inducing agent of the present invention or administered as aseparate composition to a patient in combination with theimmunity-inducing agent of the present invention.

Examples of the immunoenhancer include adjuvants. Adjuvants can enhancethe immune response by providing a reservoir of antigen (extracellularlyor within macrophages), activating macrophages and stimulating specificsets of lymphocytes, thereby enhancing the immune response and hence theanticancer action. Therefore, especially in cases where theimmunity-inducing agent of the present invention is used for therapyand/or prophylaxis of cancer, the immunity-inducing agent preferablycomprises an adjuvant, in addition to the above-described polypeptide asan effective ingredient. Many types of adjuvants are well-known in theart, and any of these adjuvants may be used. Specific examples of theadjuvants include MPL (SmithKline Beecham), homologues of Salmonellaminnesota Re 595 lipopolysaccharide obtained after purification and acidhydrolysis of the lipopolysaccharide; QS21 (SmithKline Beecham), pureQA-21 saponin purified from an extract of Quillja saponaria; DQS21described in PCT application WO96/33739 (SmithKline Beecham); QS-7,QS-17, QS-18 and QS-L1 (So and 10 colleagues, “Molecules and cells”,1997, Vol. 7, p. 178-186); Freund's incomplete adjuvant; Freund'scomplete adjuvant; vitamin E; Montanide; alum; CpG oligonucleotides(see, for example, Kreig and 7 colleagues, Nature, Vol. 374, p.546-549); poly-I:C and derivatives thereof (e.g., poly ICLC); andvarious water-in-oil emulsions prepared from biodegradable oils such assqualene and/or tocopherol. Among these, Freund's incomplete adjuvant;Montanide; poly-I:C and derivatives thereof; and CpG oligonucleotidesare preferred. The mixing ratio between the above-described adjuvant andthe polypeptide is typically about 1:10 to 10:1, preferably about 1:5 to5:1, more preferably about 1:1. However, the adjuvant is not limited tothe above-described examples, and adjuvants known in the art other thanthose described above may also be used when the immunity-inducing agentof the present invention is administered (see, for example, Goding,“Monoclonal Antibodies: Principles and Practice, 2nd edition”, 1986).Preparation methods for mixtures or emulsions of a polypeptide and anadjuvant are well-known to those skilled in the art of vaccination.

Further, in addition to the above-described adjuvants, factors thatstimulate the immune response of the subject may be used as theabove-described immunoenhancer. For example, various cytokines having aproperty to stimulate lymphocytes and/or antigen-presenting cells may beused as the immunoenhancer in combination with the immunity-inducingagent of the present invention. A number of such cytokines capable ofenhancing the immune response are known to those skilled in the art, andexamples thereof include, but are not limited to, interleukin-12(IL-12), GM-CSF, IL-18, interferon-α, interferon-β, interferon-ω,interferon-γ, and Flt3 ligand, which have been shown to enhance theprophylactic action of vaccines. Such factors may also be used as theabove-described immunoenhancer, and may be contained in theimmunity-inducing agent of the present invention, or may be prepared asa separate composition to be used in combination with theimmunity-inducing agent of the present invention, to be administered toa patient.

By bringing the above-described polypeptide into contact withantigen-presenting cells in vitro, the antigen-presenting cells can bemade to present the polypeptide. That is, the polypeptides (a) to (c)described above can be used as agents for treating antigen-presentingcells. Examples of the antigen-presenting cells which may be preferablyused include dendritic cells and B cells having MHC class I molecules.Various MHC class I molecules have been identified and are well-known.MHC molecules in human are called HLA. Examples of HLA class I moleculesinclude HLA-A, HLA-B and HLA-C, more specifically, HLA-A1, HLA-A0201,HLA-A0204, HLA-A0205, HLA-A0206, HLA-A0207, HLA-A11, HLA-A24, HLA-A31,HLA-A6801, HLA-B7, HLA-B8, HLA-B2705, HLA-B37, HLA-Cw0401 andHLA-Cw0602.

The dendritic cells or B cells having MHC class I molecules can beprepared from peripheral blood by a well-known method. For example,tumor-specific dendritic cells can be induced by inducing dendriticcells from bone marrow, umbilical cord blood or patient's peripheralblood using granulocyte-macrophage colony-stimulating factor (GM-CSF)and IL-3 (or IL-4), and then adding a tumor-related peptide to theculture system.

By administering an effective amount of such dendritic cells, a responsedesired for therapy of a cancer can be induced. As the cells to be used,bone marrow or umbilical cord blood donated by a healthy individual, orbone marrow, peripheral blood or the like from the patient himself maybe used. When autologous cells of the patient are used, high safety canbe attained and serious side effects are expected to be avoided. Theperipheral blood or bone marrow may be any of a fresh sample,cold-stored sample and frozen sample. As for the peripheral blood, wholeblood may be cultured or the leukocyte components alone may be separatedand cultured, and the latter is more efficient and thus preferred.Further, among the leukocyte components, mononuclear cells may beseparated. In cases where the cells are originated from bone marrow orumbilical cord blood, the whole cells constituting the bone marrow maybe cultured, or mononuclear cells may be separated therefrom andcultured. Peripheral blood, the leukocyte components thereof and bonemarrow cells contain mononuclear cells, hematopoietic stem cells andimmature dendritic cells, from which dendritic cells are originated, andalso CD4-positive cells and the like. As for the cytokine to be used,the production method thereof is not restricted, and anaturally-occurring or recombinant cytokine or the like may be employedas long as its safety and physiological activity have been confirmed.Preferably, a preparation with assured quality for medical use is usedin a minimum necessary amount. The concentration of the cytokine(s) tobe added is not restricted as long as the dendritic cells are induced atthe concentration, and usually, the total concentration of thecytokine(s) is preferably about 10 to 1000 ng/mL, more preferably about20 to 500 ng/mL. The culture may be carried out using a well-knownmedium usually used for culture of leukocytes. The culturing temperatureis not restricted as long as proliferation of the leukocytes is attainedat the temperature, and a temperature of about 37° C., which is the bodytemperature of human, is most preferred. The atmospheric environmentduring the culturing is not restricted as long as proliferation of theleukocytes is attained under the environment, and 5% CO₂ is preferablyallowed to flow. The culturing period is not restricted as long as anecessary number of the cells are induced therewith, and usually 3 daysto 2 weeks. As for the apparatuses used for separation and culturing ofthe cells, appropriate apparatuses, preferably those whose safety uponapplication to medical uses have been confirmed and whose operations arestable and simple, may be employed. In particular, as for thecell-culturing apparatus, not only a general vessel such as a Petridish, flask or bottle, but also a layer type vessel, multistage vessel,roller bottle, spinner type bottle, bag type culturing vessel, hollowfiber column or the like may be used.

The method per se to be used for bringing the above-describedpolypeptide into contact with the antigen presenting cells in vitro maybe carried out by a well-known method. For example, it may be carriedout by culturing the antigen-presenting cells in a culture mediumcontaining the above-described polypeptide. The concentration of thepeptide in the medium is not restricted, and usually about 1 to 100μg/ml, preferably about 5 to 20 μg/ml. The cell density during theculture is not restricted and usually about 10³ to 10⁷ cells/ml,preferably about 5×10⁴ to 5×10⁶ cells/ml. The culture may be carried outaccording to a conventional method at 37° C. under the atmosphere of 5%CO₂. The maximum length of the peptide which can be presented on thesurface of the antigen-presenting cells is usually about 30 amino acidresidues. Therefore, in cases where the antigen-presenting cells arebrought into contact with the polypeptide in vitro, the polypeptide maybe prepared such that its length is not more than about 30 amino acidresidues, although the length is not restricted.

By culturing the antigen-presenting cells in the coexistence of theabove-described polypeptide, the polypeptide is incorporated into an MHCmolecule of the antigen-presenting cells and presented on the surface ofthe antigen-presenting cells. Therefore, using the above-describedpolypeptide, isolated antigen-presenting cells containing the complexbetween the polypeptide and the MHC molecule can be prepared. Suchantigen-presenting cells can present the polypeptide against T cells invivo or in vitro, and thereby induce, and allow proliferation of,cytotoxic T cells specific to the polypeptide.

By bringing the thus prepared antigen-presenting cells having thecomplex between the above-described polypeptide and the MHC moleculeinto contact with T cells in vitro, cytotoxic T cells specific to thepolypeptide can be induced and allowed to proliferate. This may becarried out by coculturing the above-described antigen-presenting cellsand T cells in a liquid medium. For example, it may be attained bysuspending the antigen-presenting cells in a liquid medium, placing thesuspension in vessels such as wells of a microplate, adding T cellsthereto and then culturing the cells. The mixing ratio of theantigen-presenting cells with respect to the T cells in the coculture isnot restricted, and is usually about 1:1 to 1:100, preferably about 1:5to 1:20 in terms of the ratio between the numbers of the cells. Thedensity of the antigen-presenting cells to be suspended in the liquidmedium is not restricted, and is usually about 100 to 10,000,000cells/ml, preferably about 10,000 to 1,000,000 cells/ml. The cocultureis preferably carried out in accordance with a conventional method at37° C. under the atmosphere of 5% CO₂. The culturing period is notrestricted, and is usually 2 days to 3 weeks, preferably about 4 days to2 weeks. The coculture is preferably carried out in the presence of oneor more interleukins such as IL-2, IL-6, IL-7 and/or IL-12. In suchcases, the concentration of IL-2 or IL-7 is usually about 5 to 20 U/ml,the concentration of IL-6 is usually about 500 to 2000 U/ml, and theconcentration of IL-12 is usually about 5 to 20 ng/ml, but theconcentrations of the interleukins are not restricted thereto. The abovecoculture may be repeated once to several times with addition of freshantigen-presenting cells. For example, the operation of discarding theculture supernatant after the coculture and adding a fresh suspension ofantigen-presenting cells to further conduct the coculture may berepeated once to several times. The conditions of each coculture may bethe same as described above.

By the above-described coculture, cytotoxic T cells specific to thepolypeptide are induced and allowed to proliferate. Thus, using theabove-described polypeptide, isolated T cells can be prepared whichselectively bind to the complex between the polypeptide and the MHCmolecule.

As described in the Examples below, the gene encoding PDS5A is expressedspecifically in breast cancer cells, breast cancer tissues, brain tumorcells, brain tumor tissues, esophagus cancer cells, esophagus cancertissues, lung cancer cells, lung cancer tissues, renal cancer cells,renal cancer tissues, colon cancer cells, colon cancer tissues, perianaladenocarcinoma tissues, perianal adenocarcinoma cells, neuroblastomacells and leukemia cells. Therefore, it is thought that, in these cancerspecies, a significantly larger amount of PDS5A exists than in normalcells. When cytotoxic T cells prepared as described above areadministered to a living body while a part of PDS5A existing in cancercells is presented by MHC molecules on the surface of the cancer cells,the cytotoxic T cells can damage the cancer cells using the presentedpolypeptide as a marker. Since antigen-presenting cells presenting theabove-described polypeptide can induce, and allow proliferation of,cytotoxic T cells specific to the polypeptide also in vivo, cancer cellscan be damaged also by administering the antigen-presenting cells to aliving body. That is, the cytotoxic T cells and the antigen-presentingcells prepared using the polypeptide are also effective as therapeuticand/or prophylactic agents for cancer, similarly to theimmunity-inducing agent of the present invention.

In cases where the above-described isolated antigen-presenting cells orisolated T cells are administered to a living body, these are preferablyprepared by treating antigen presenting cells or T cells collected fromthe patient to be treated with the polypeptide (a) to (c) as describedabove in order to avoid the immune response in the living body thatattacks these cells as foreign bodies.

The therapeutic and/or prophylactic agent for cancer comprising as aneffective ingredient the antigen-presenting cells or T cells ispreferably administered via a parenteral administration route, forexample, by intravenous or intraarterial administration. The dose isappropriately selected depending on the symptoms, the purpose ofadministration and the like, and is usually 1 cell to 10,000,000,000,000cells, preferably 1,000,000 cells to 1,000,000,000 cells, which dose ispreferably administered once every several days to once every severalmonths. The formulation may be, for example, the cells suspended inphysiological buffered saline, and the formulation may be used incombination with another/other anticancer preparation(s) and/orcytokine(s). Further, one or more additives well-known in the field offormulation of pharmaceuticals may also be added.

Also by expressing a polynucleotide encoding any of the polypeptides (a)to (c) in the body of the subject animal, antibody production andcytotoxic T cells can be induced in the living body, and an effectcomparable to that obtained in the case of administration of thepolypeptide can be obtained. That is, the immunity-inducing agent of thepresent invention may be one comprising as an effective ingredient arecombinant vector having a polynucleotide encoding any of thepolynucleotides (a) to (c), which recombinant vector is capable ofexpressing the polypeptide in a living body. Such a recombinant vectorcapable of expressing an antigenic polypeptide as shown in thelater-mentioned Examples is also called a gene vaccine.

The vector used for production of the gene vaccine is not restricted aslong as it is a vector capable of expressing the polypeptide in a cellof the subject animal (preferably in a mammalian cell), and may beeither a plasmid vector or a virus vector, and any known vector in thefield of gene vaccines may be used. The polynucleotide such as DNA orRNA encoding the above-described polypeptide can be easily prepared asmentioned above by a conventional method. Incorporation of thepolynucleotide into the vector can be carried out using a methodwell-known to those skilled in the art.

The administration route of the gene vaccine is preferably a parenteralroute such as intramuscular, subcutaneous, intravenous or intraarterialadministration, and the dose may be appropriately selected depending onthe type of the antigen and the like, and is usually about 0.1 μg to 100mg, preferably about 1 μg to 10 mg in terms of the weight of the genevaccine per 1 kg of body weight.

Examples of the method using a virus vector include those wherein apolynucleotide encoding the above-described polypeptide is incorporatedinto an RNA virus or DNA virus, such as a retrovirus, adenovirus,adeno-associated virus, herpes virus, vaccinia virus, pox virus,poliovirus or Sindbis virus, and then a subject animal is infected withthe resulting virus. Among these methods, those using a retrovirus,adenovirus, adeno-associated virus, vaccinia virus or the like areespecially preferred.

Examples of other methods include a method wherein an expression plasmidis directly intramuscularly administered (DNA vaccine method), liposomemethod, lipofectin method, microinjection method, calcium phosphatemethod and electroporation method, and the DNA vaccine method andliposome method are especially preferred.

Methods for actually making the gene encoding the above-describedpolypeptide used in the present invention act as a pharmaceuticalinclude the in vivo method wherein the gene is directly introduced intothe body, and the ex vivo method wherein a certain kind of cells arecollected from a subject animal and the gene is introduced into thecells ex vivo, followed by returning the cells to the body (NikkeiScience, 1994, April, p. 20-45; The Pharmaceutical Monthly, 1994, Vol.36, No. 1, p. 23-48; Experimental Medicine, Extra Edition, 1994, Vol.12, No. 15; and references cited in these literatures, and the like).The in vivo method is more preferred.

In cases where the gene is administered by the in vivo method, the genemay be administered through an appropriate administration routedepending on the disease to be treated, symptoms and so on. It may beadministered by, for example, intravenous, intraarterial, subcutaneousor intramuscular administration. In cases where the gene is administeredby the in vivo method, the gene may be formulated into a preparationsuch as a solution, and in general, it is formulated into an injectionsolution or the like containing DNA encoding the above-described peptideof the present invention as an effective ingredient. A commonly usedcarrier(s) may be also added thereto as required. In the case of aliposome or membrane fusion liposome (Sendai virus (HVJ)-liposome or thelike) containing the DNA, the liposome may be formulated into a liposomepreparation such as a suspension, frozen preparation or centrifugallyconcentrated frozen preparation.

In the present invention, “the base sequence shown in SEQ ID NO:1”includes not only the base sequence expressly written in SEQ ID NO:1,but also the sequence complementary thereto. Thus, “the polynucleotidehaving the base sequence shown in SEQ ID NO:1” includes asingle-stranded polynucleotide having the base sequence expresslywritten in SEQ ID NO:1, a single-stranded polynucleotide having the basesequence complementary thereto, and a double-stranded polynucleotidecomposed of these single-stranded polynucleotides. When a polynucleotideencoding the polypeptide used in the present invention is prepared, anyone of these base sequences is appropriately selected, and those skilledin the art can easily carry out the selection.

Further, since the polypeptide used in the present invention isexpressed specifically in cancer, the polypeptide specifically reactsonly with the serum in a cancer-bearing living body, so that thepolypeptide of the present invention is used also for detection ofcancer.

In the above-described method for detecting cancer, a sample separatedfrom a living body is used to measure expression of a polypeptide havingany one of the amino acid sequences shown in SEQ ID NOs:2, 4, 6, 8, 10,12 and 44, or a polypeptide as a homologous factor thereof, having asequence identity of not less than 90%, preferably not less than 95%,more preferably not less than 98%, still more preferably not less than99% or not less than 99.5% to the polypeptide. Examples of the methodfor measuring the expression of the polypeptide using the sampleincludes a method in which an antibody against the polypeptide, whichantibody is contained in the sample, is measured by immunoassay (Method1); a method in which the polypeptide per se contained in the sample ismeasured by immunoassay (Method 2); and a method in which mRNA containedin the sample and encoding the polypeptide is measured (Method 3). Inthe method of the present invention, the expression of the polypeptidemay be measured by any of these methods. In the present invention, theterm “measurement” includes detection, quantification andsemi-quantification.

Here, PDS5A is a polypeptide identified, by the SEREX method using acanine breast cancer-derived cDNA library and serum obtained from thesame patient dog, as a polypeptide that binds to an antibodyspecifically existing in the serum derived from the tumor-bearing dog(cancer-specific antibody) (see Example 1). That is, in the living bodyof the tumor-bearing dog, an antibody against PDS5A is specificallyinduced. Therefore, by measuring the antibody against PDS5A in theliving body of the tumor-bearing dog, a cancer expressing PDS5A can alsobe detected. Further, also by measuring PDS5A as an antigen by Method 2,the canine cancer can be detected. Further, since, as described in thelater-mentioned Examples, mRNA encoding the antigen polypeptide isexpressed at significantly higher levels in cancer cells and cancertissues, especially in breast cancer cells, breast cancer tissues, braintumor cells, brain tumor tissues, esophagus cancer cells, esophaguscancer tissues, lung cancer cells, lung cancer tissues, renal cancercells, renal cancer tissues, colon cancer cells, colon cancer tissues,perianal adenocarcinoma cells, perianal adenocarcinoma tissues,neuroblastoma cells and leukemia cells, compared to the normal tissues(see Example 1), the canine cancer can be detected also by measuring themRNA.

In Method 1 above, measurement of the cancer-specific antibody which mayexist in the sample can be easily carried out by immunoassay using anantigenic substance which undergoes antigen-antibody reaction with theantibody. The immunoassay per se is a conventional well-known method asexplained in detail below. Examples of the antigenic substance which maybe used in the immunoassay include the polypeptides (a) to (c). Sinceantibodies have cross-reactivity, even a molecule other than theantigenic substance corresponding to the original immunogen may be boundto an antibody induced against the immunogen by antigen-antibodyreaction, as long as the molecule has a structure thereon similar to anepitope of the immunogen. For example, polypeptides having a highsequence identity therebetween often have similar epitope structures,and, in this case, the both polypeptides may have the same antigenicity.As concretely described in the Examples below, the human-derivedpolypeptide of SEQ ID NO:4 or 44 undergoes antigen-antibody reactionwith the above-described antibody induced in the body of acancer-bearing dog. Therefore, in Method 1 of the present invention, anymammalian homologous factor may be used as the antigen in theimmunoassay.

An antigenic substance having a complex structure and a large molecularweight, such as a protein, usually has a plurality of sites havingdifferent structures on the molecule. Therefore, against such anantigenic substance, a plurality of kinds of antibodies which recognizethe respective plurality of sites are produced in a living body. Thatis, an antibody induced in a living body against an antigenic substancesuch as a protein is a polyclonal antibody, which is a mixture of aplurality of kinds of antibodies. It should be noted that, in thepresent invention, the term “polyclonal antibody” means antibodies whichexist in serum from a living body having an antigenic substance thereinand were induced in the living body against the antigenic substance.

Measurement of the antibody in a sample may easily be carried out byimmunoassay using the above-described polypeptide as an antigen.Immunoassays per se are well-known in the art, and include, whenclassified based on the reaction mode, the sandwich method, competitionmethod, agglutination method, Western blotting and the like. Whenclassified based on the label, immunoassays include radioimmunoassay,fluorescence immunoassay, enzyme immunoassay, biotin immunoassay and thelike, and the immunoassay of the above-described antibody may be carriedout by any of these immunoassays. Although not restricted, the sandwichELISA and the agglutination method may be preferably applied as themethod of immunoassay of the above antibody in the present invention,since the operations are simple and a large-scale apparatus is notnecessary in these methods. In cases where an enzyme is used as thelabel of the antibody, the enzyme is not particularly restricted as longas it satisfies conditions such as a large turnover number, stabilityupon binding with the antibody, and specific coloring of the substrate,and examples of the enzyme which may be used include enzymes used in anordinary enzyme immunoassay, such as peroxidase, β-galactosidase,alkaline phosphatase, glucose oxidase, acetylcholinesterase,glucose-6-phosphate dehydrogenase, and malate dehydrogenase. An enzymeinhibitor, coenzyme and/or the like may also be used. Binding of theenzyme with the antibody may be carried out by a known method using across-linking agent such as a maleimide compound. As a substrate, aknown substance may be used depending on the type of the enzyme to beused. For example, in cases where peroxidase is used as the enzyme,3,3′,5,5′-tetramethylbenzidine may be used; and in cases where alkalinephosphatase is used as the enzyme, para-nitrophenol or the like may beused. As a radioisotope, one used in an ordinary radioimmunoassay, suchas ¹²⁵I or ³H may be used. As a fluorescent dye, one used in an ordinaryfluorescent antibody technique, such as fluorescein isothiocyanate(FITC), tetramethylrhodamine isothiocyanate (TRITC) or the like may beused.

These immunoassays per se are well-known in the art and do not need tobe explained in the present specification. Briefly, in a sandwichimmunoassay, for example, the above-mentioned polypeptide used as anantigen is immobilized on a solid phase and then reacted with a samplesuch as a serum. After washing the solid phase, the resultant is reactedwith an appropriate secondary antibody. After washing the solid phase,the secondary antibody bound to the solid phase is measured. This methodis preferred as an embodiment of the method of the present invention fordetecting cancer since, in this method, immobilization of the antigenpolypeptide to the solid phase enables simple removal of unboundsecondary antibodies. As the secondary antibody, an anti-dog IgGantibody may be used in cases where, for example, the sample is derivedfrom a dog. By preliminarily labeling the secondary antibody with alabeling substance exemplified above, the secondary antibody bound tothe solid phase can be measured. The thus measured amount of thesecondary antibody corresponds to the amount of the above-mentionedantibody in the serum sample. In cases where an enzyme is used as thelabeling substance, the amount of the antibody may be measured by addinga substrate which develops a color upon decomposition by an enzymaticactivity, and then optically measuring the amount of decomposition ofthe substrate. In cases where a radioisotope is used as the labelingsubstance, the amount of radiation emitted from the radioisotope may bemeasured with a scintillation counter or the like.

In Method 2 of the present invention, the polypeptide of SEQ ID NO:2, 4,6, 8, 10, 12 or 44 or a homologous factor thereof, which may becontained in a sample obtained from a living body is measured. Asmentioned above, the amount of a cancer-specific antibody whichundergoes antigen-antibody reaction with the polypeptide of SEQ ID NO:2,4, 6, 8, 10, 12 or 44 or a homologous factor thereof is significantlylarger in cancer patients, and this indicates that the amount ofproduction of the polypeptide or a homologous factor thereof, whichcorresponds to an antigen of the cancer-specific antibody, issignificantly larger in the cancer patients. Therefore, cancer in aliving body can be detected also by measuring the polypeptide of SEQ IDNO:2, 4, 6, 8, 10, 12 or 44 or a homologous factor thereof similarly toMethod 1 described above.

The polypeptide in a sample can be easily measured by a well-knownimmunoassay. More particularly, for example, by preparing an antibody oran antigen-binding fragment thereof which undergoes antigen-antibodyreaction with the polypeptide shown in SEQ ID NO:2, 4, 6, 8, 10, 12 or44 and using this in an immunoassay, the polypeptide having the sequenceshown in SEQ ID NO:2, 4, 6, 8, 10, 12 or 44 or a homologous factorthereof which may exist in the sample can be measured. The immunoassayper se is a well-known conventional method as described above.

The term “antigen-binding fragment” herein means an antigen fragmentsuch as the Fab fragment or F(ab′)2 fragment contained in the antibodymolecule, which has a binding capacity to an antigen. The antibody maybe either a polyclonal antibody or monoclonal, and a monoclonal antibodyis preferred in an immunoassay and the like because a highreproducibility can be obtained therewith. The methods of preparation ofa polyclonal antibody and a monoclonal antibody using a polypeptide asan immunogen are well known, and can be easily carried out byconventional methods. For example, antibodies against a polypeptide canbe induced by immunizing an animal with, as an immunogen, thepolypeptide conjugated to a carrier protein such as keyhole limpethemocyanin (KLH) or casein, together with an adjuvant.Antibody-producing cells such as spleen cells or lymphocytes are thencollected from the immunized animal and fused with myeloma cells toprepare hybridomas. Among the hybridomas, one producing an antibodywhich binds to the polypeptide shown in SEQ ID NO:2, 4, 6, 8, 10, 12 or44, or a homologous factor thereof is selected and proliferated, andthen a monoclonal antibody whose corresponding antigen is theabove-mentioned protein can be obtained from the culture supernatant.The above-described method is a conventional well-known method.

In Method 3 of the present invention, mRNA which may be contained in asample obtained from a living body and encodes PDS5A is measured. Asconcretely shown in the Examples below, mRNA encoding PDS5A issignificantly highly expressed in tissues and cells of cancer, breastcancer, brain tumor, esophagus cancer, lung cancer, renal cancer, coloncancer, perianal adenocarcinoma, neuroblastoma and leukemia. Therefore,also by measuring the mRNA in the sample, cancer in the living body canbe detected.

In the detection method of the present invention, whether or not asubject living body is suffering from cancer is judged based on theexpression level of the polypeptide measured as described above.Although the cancer detection may be attained simply by measuringexpression of the polypeptide in the subject living body, it ispreferred, from the viewpoint of enhancement of the detection accuracy,to obtain a normal reference value by investigating the expression levelof the polypeptide (the amount of the antibody, polypeptide or mRNA) inone or more samples from healthy individuals, followed by comparison ofthe measured value in the subject living body with the normal referencevalue. In cases where a higher detection accuracy is required, a cancerreference value may be obtained by investigating the expression level ofthe polypeptide in samples obtained from many patients known to besuffering from cancer, followed by comparison of the measured value inthe subject living body both with the normal reference value and withthe cancer reference value. The reference values may be determined by,for example, digitizing the expression level of the polypeptide in eachsample and calculating the mean value. The normal reference value andthe cancer reference value may be preliminarily determined byinvestigating the expression level of the polypeptide in many healthyindividuals and cancer patients. Thus, in cases where comparison withthe reference value(s) is carried out in the method of the presentinvention, a preliminarily determined reference value(s) may be used.

The detection method of the present invention may be used in combinationwith detection with another cancer antigen or cancer marker. By this,the accuracy of detection of cancer can be further increased.

By the detection method of the present invention, cancers in a livingbody can be detected. By the method of the present invention, even aninvisible small cancer or a cancer which exists in a deep part of a bodycan be detected, and thus the method is useful for early detection ofcaners. Further, by applying the detection method of the presentinvention to patients in the follow-up period after cancer therapy, arecurrent cancer, if any, can be detected at an early stage.

In a tumor-bearing living body, as the number of cancer cells expressingthe specific polypeptide to be measured in the present inventionincreases, the amounts of accumulation of the polypeptide and the mRNAencoding it in the living body increase, leading to increased productionof antibodies against the polypeptide in the serum. On the other hand,as the number of cancer cells decreases, the amounts of accumulation ofthe polypeptide and the mRNA encoding it in the living body decrease,leading to decrease in antibodies against the polypeptide in the serum.Thus, in cases where the expression level of the specific polypeptide ishigh, it can be determined that tumor growth and/or metastasis of canceroccurred, that is, the stage of progression of cancer is advanced.

Further, as shown in the Examples below, when compared between the samekind of tumors, a malignant one produces a significantly higher amountof the antibodies than a benign one. Therefore, in cases where theexpression level of the specific polypeptides is high, it can bedetermined that the grade of cancer malignancy is high. That is, thegrade of cancer malignancy can also be detected by the method of thepresent invention.

Furthermore, the effect of a cancer therapy can be monitored based onincrease or decrease of the expression level of the specificpolypeptide. Therefore, by observing the expression level of theabove-mentioned polypeptide in an individual during or after a cancertherapy, one can obtain a clue(s) to know the effect of an anticancerdrug, presence/absence of a residual tumor after extirpation of thetumor, and/or, even during the follow-up, metastasis and/or recurrence,as early as possible. In cases where a therapy is/was appropriate, theexpression level of the polypeptide becomes lower than that in thepatient in the tumor-bearing state before the therapy, and therefore theeffect of the therapy that was (or is being) provided for the livingbody can be judged to have been (or to be) excellent. In cases where theexpression level of the polypeptide increased or is maintained, or incases where the expression level once decreased and then increasedagain, the therapeutic effect can be judged to be insufficient, and thisobservation can be a useful basis for selection of a therapeutic method,such as use of another therapeutic method or alteration of the dose ofan anti-cancer agent.

Preferred examples of the cancer as the subject of the method fordetecting cancer of the present invention include cancers expressingPDS5A, such as breast cancer, brain tumor, esophagus cancer, lungcancer, renal cancer, colon cancer, perianal adenocarcinoma,neuroblastoma and leukemia. The living body as the subject of the methodof the present invention is preferably a mammal, more preferably human,dog or cat.

The sample to be provided for the method of the present inventioninclude body fluids such as blood, serum, plasma, ascites and pleuraleffusion, and tissues and cells. Particularly, serum, plasma, ascitesand pleural effusion may be preferably used in Method 1 and Method 2above. A tissue sample and cell sample are preferred in the case ofMethod 3 above in which mRNA is measured.

The polypeptide used as the antigen for the immunoassay in Method 1 maybe provided as a reagent for cancer detection. The reagent may consistessentially of the above-mentioned polypeptide, or may contain, forexample, various additives useful for stabilizing the polypeptide,and/or the like. The reagent may be provided also in a state where it isimmobilized on a solid phase such as a plate or membrane.

When the polypeptide shown in SEQ ID NO:2, 4, 6, 8, 10, 12 or 44 or ahomologous factor thereof is to be immunoassayed in Method 2, anantibody or an antigen-binding fragment thereof which undergoesantigen-antibody reaction with the polypeptide or a homologous factorthereof may also be provided as a reagent for cancer detection. Also inthis case, the reagent for cancer detection may consist essentially ofthe antibody or antigen-binding fragment, or may contain, for example,various additives useful for stabilizing the antibody or antigen-bindingfragment, and/or the like. The antibody or antigen-binding fragment mayalso be in a state where a metal such as manganese or iron is boundthereto. Administration of such a metal-bound antibody orantigen-binding fragment into a living body causes higher accumulationof the antibody or antigen-binding fragment at locations where theantigen protein exists in a larger amount, so that measurement of themetal by MRI or the like enables detection of existence of cancer cellsthat produces the antigen protein.

Furthermore, the above-described polynucleotide for cancer detection tobe used for measuring mRNA in Method 3 may also be provided as a reagentfor cancer detection. Also in this case, the reagent for cancerdetection may consist essentially of the polynucleotide, or may contain,for example, various additives useful for stabilizing thepolynucleotide, and/or the like. The polynucleotide for cancer detectioncontained in the reagent is preferably a primer or a probe.

EXAMPLES

The present invention will now be described more concretely by way ofExamples.

Example 1 Obtaining Novel Cancer Antigen Protein by SEREX Method

(1) Preparation of cDNA Library

Total RNA was extracted from a breast cancer tissue of a tumor-bearingdog by the Acid guanidium-Phenol-Chloroform method, and poly(A) RNA waspurified using Oligotex-dT30 mRNA purification Kit (manufactured byTakara Shuzo Co., Ltd.) in accordance with the protocol attached to thekit.

Using the obtained mRNA (5 μg), a cDNA phage library was synthesized.For the preparation of the cDNA phage library, cDNA Synthesis Kit,ZAP-cDNA Synthesis Kit, and ZAP-cDNA Gigapack III Gold Cloning Kit(manufactured by STRATAGENE) were used in accordance with the protocolsattached to the kits. The size of the prepared cDNA phage library was1×10⁶ pfu/ml.

(2) Screening of cDNA Library with Serum

Using the prepared cDNA phage library, immunoscreening was carried out.More particularly, the host E. coli (XL1-Blue MRF′) was infected withthe library such that 2340 clones appear on an NZY agarose plate havinga size of Φ90×15 mm, and cultured at 42° C. for 3 to 4 hours to allowthe phage to form plaques. The plate was covered with a nitrocellulosemembrane (Hybond C Extra: manufactured by GE Healthcare Bio-Science)impregnated with IPTG (isopropyl-β-D-thiogalactoside) at 37° C. for 4hours to allow induction and expression of proteins, which were thustransferred to the membrane. Subsequently, the membrane was recoveredand soaked in TBS (10 mM Tris-HCl, 150 mM NaCl; pH 7.5) containing 0.5%non-fat dry milk, followed by shaking it at 4° C. overnight to suppressnon-specific reactions. This filter was then allowed to react with500-fold diluted canine patient serum at room temperature for 2 to 3hours.

As the above-described canine patient serum, serum collected from acanine patient suffering from perianal tumor was used. The serum wasstored at −80° C. and pretreated immediately before use. The method ofthe pretreatment of the serum was as follows. That is, the host E. coli(XL1-Blue MRF′) was infected with λ ZAP Express phage to which noforeign gene was inserted, and then cultured on NZY plate medium at 37°C. overnight. Subsequently, 0.2 M NaHCO₃ buffer (pH 8.3) containing 0.5M NaCl was added to the plate, and the plate was left to stand at 4° C.for 15 hours, followed by collecting the supernatant as an E. coli/phageextract. Thereafter, the collected E. coli/phage extract was allowed toflow through an NHS column (manufactured by GE Healthcare Bio-Science)to immobilize proteins derived from the E. coli/phage thereon. The serumfrom the canine patient was allowed to flow through and react with thisprotein-immobilized column to remove antibodies adsorbed to E. coliand/or the phage. The serum fraction that passed through the column was500-fold diluted with TBS containing 0.5% non-fat dry milk, and theresulting diluent was used as the material for the immunoscreening.

The membrane on which the thus treated serum and the above-describedfusion protein were blotted was washed 4 times with TBS-T (0.05% Tween20/TBS), and allowed to react with goat anti-dog IgG (Goat anti DogIgG-h+I HRP conjugated: manufactured by BETHYL Laboratories) 5,000-folddiluted with TBS containing 0.5% non-fat dry milk as a secondaryantibody at room temperature for 1 hour, followed by detection by theenzyme coloring reaction using the NBT/BCIP reaction solution(manufactured by Roche). Colonies at positions where a positive coloringreaction was observed were recovered from the NZY agarose plate having asize of Φ90×15 mm, and dissolved in 500 μl of SM buffer (100 mM NaCl, 10mM MgClSO₄, 50 mM Tris-HCl, 0.01% gelatin; pH 7.5). The screening wasrepeated as a second and third screening in the same manner as describedabove until a single coloring reaction-positive colony was obtained,thereby isolating one positive clone after screening of 30940 phageclones reactive with IgG in the serum.

(3) Sequence Homology Search of Isolated Antigen Gene

To subject the single positive clone isolated by the above-describedmethod to a base sequence analysis, an operation of conversion of thephage vector to a plasmid vector was carried out. More particularly, 200μl of a solution prepared such that the host E. coli (XL1-Blue MRF′) wascontained at an absorbance OD₆₀₀ of 1.0 was mixed with 100 μl of apurified phage solution and further with 1 μl of ExAssist helper phage(manufactured by STRATAGENE), and the reaction was allowed to proceed at37° C. for 15 minutes. To the reaction mixture, 3 ml of LB medium wasadded, and the resulting mixture was cultured at 37° C. for 2.5 to 3hours, followed by immediate incubation in a water bath at 70° C. for 20minutes. The mixture was then centrifuged at 4° C. at 1,000 xg for 15minutes, and the supernatant was recovered as a phagemid solution.Subsequently, 200 μl of a solution prepared such that the phagemid hostE. coli (SOLR) was contained at an absorbance OD₆₀₀ of 1.0 was mixedwith 10 μl of a purified phage solution, and the reaction was allowed toproceed at 37° C. for 15 minutes. Thereafter, 50 μl of the reactionmixture was plated on LB agar medium supplemented with ampicillin (finalconcentration: 50 μg/ml), and cultured at 37° C. overnight. A singlecolony of transformed SOLR was recovered and cultured in LB mediumsupplemented with ampicillin (final concentration: 50 μg/ml) at 37° C.,followed by purification of plasmid DNA having the insert of interestusing QIAGEN plasmid Miniprep Kit (manufactured by Qiagen).

The purified plasmid was subjected to analysis of the full-lengthsequence of the insert by the primer walking method using the T3 primerdescribed in SEQ ID NO:13 and the T7 primer described in SEQ ID NO:14.By this sequence analysis, the gene sequence described in SEQ ID NO:1was obtained. Using the base sequence and the amino acid sequence ofthis gene, homology search against known genes was carried out using asequence homology search program BLAST(http://www.ncbi.nlm.nih.gov/BLAST/). As a result, it was revealed thatthe obtained gene is the PDS5A gene. Human PDS5A, which is a humanhomologous factor of canine PDS5A, had a sequence identity of 94% interms of the base sequence and 99% in terms of the amino acid sequence;murine PDS5A, which is a murine homologous factor, had a sequenceidentity of 91% in terms of the base sequence and 99% in terms of theamino acid sequence; bovine PDS5A, which is a bovine homologous factor,had a sequence identity of 95% in terms of the base sequence and 99% interms of the amino acid sequence; equine PDS5A, which is a equinehomologous factor, had a sequence identity of 96% in terms of the basesequence and 99% in terms of the amino acid sequence; and chicken PDS5A,which is a chicken homologous factor, had a sequence identity of 83% interms of the base sequence and 98% in terms of the amino acid sequence.In terms of human PDS5A, the base sequence is shown in SEQ ID NOs:3 and43, and the amino acid sequence is shown in SEQ ID NOs:4 and 44; interms of murine PDS5A, the base sequence is shown in SEQ ID NO:5, andthe amino acid sequence is shown in SEQ ID NO:6; in terms of bovinePDS5A, the base sequence is shown in SEQ ID NO:7, and the amino acidsequence is shown in SEQ ID NO:8; in terms of equine PDS5A, the basesequence is shown in SEQ ID NO:9, and the amino acid sequence is shownin SEQ ID NO:10; and in terms of chicken PDS5A, the base sequence isshown in SEQ ID NO:11, and the amino acid sequence is shown in SEQ IDNO:12.

(4) Analysis of Expression in Various Tissues

Expression of the genes obtained by the above method in canine, humanand murine normal tissues and various cell lines were investigated bythe RT-PCR (Reverse Transcription-PCR) method. The reverse transcriptionreaction was carried out as follows. That is, from 50 to 100 mg of eachtissue or 5×10⁶ to 10×10⁶ cells of each cell line, total RNA wasextracted using the TRIZOL reagent (manufactured by INVITROGEN)according to the protocol described in the attached instructions. Usingthis total RNA, cDNA was synthesized with the Superscript First-StrandSynthesis System for RT-PCR (manufactured by INVITROGEN) according tothe protocol described in the attached instructions. As the cDNAs ofhuman normal tissues (brain, hippocampus, testis, colon and placenta),Gene Pool cDNA (manufactured by INVITROGEN), QUICK-Clone cDNA(manufactured by CLONETECH) and Large-Insert cDNA Library (manufacturedby CLONETECH) were used. The PCR reaction was carried out usinggene-specific primers (the canine primers described in SEQ ID NOs:15 and16, the human primers described in SEQ ID NOs:17 and 18, and the murineprimers described in SEQ ID NOs:19 and 20) as described below. That is,reagents and an attached buffer were mixed such that 0.25 μl of thesample prepared by the reverse transcription reaction, 2 μM each of theabove primers, 0.2 mM each of dNTPs, and 0.65 U ExTaq polymerase(manufactured by Takara Shuzo Co., Ltd.) were contained in a totalvolume of 25 μl, and the reaction was carried out by repeating 30 timesthe cycle of 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for1 minute using a Thermal Cycler (manufactured by BIO RAD). As a controlfor comparison, primers specific to GAPDH (the canine and human GAPDHprimers are shown in SEQ ID NOs:21 and 22; and the murine GAPDH primersare shown in SEQ ID NOs:23 and 24) were used at the same time. As aresult, as shown in FIG. 1, in terms of the canine PDS5A gene,expression was not observed in most of the healthy canine tissues, whilestrong expression was observed in the canine tumor tissues. Also interms of the human and murine PDS5A genes, expression was not observedin most of the healthy human and murine tissues, while expression wasdetected in most of the cancer cell lines (FIGS. 2 and 3), as in thecase of the canine PDS5A gene.

Example 2 Analysis of Cancer Antigenicity and Evaluation ofPharmacological Effect of PDS5A in Living Body

(1) Preparation of Recombinant Vector that Expresses PDS5A in LivingBody

Based on the base sequence of SEQ ID NO:5, a recombinant vector thatexpresses PDS5A in a living body was prepared. Reagents and an attachedbuffer were mixed together such that 1 μl of the cDNA prepared from themurine cancer cell line N2a (purchased from ATCC), which showedexpression in Example 1, 0.4 μM each of two kinds of primers having theNotI and XhoI restriction sites (shown in SEQ ID NOs:25 and 26), 0.2 mMdNTP and 1.25 U PrimeSTAR HS polymerase (manufactured by Takara ShuzoCo., Ltd.) were contained in a total volume of 50 μl, and PCR wascarried out by repeating 30 times the cycle of 98° C. for 10 seconds,55° C. for 15 seconds and 72° C. for 4 minute using a Thermal Cycler(manufactured by BIO RAD). The above-described two kinds of primers werethose for amplification of the region encoding the full-length of theamino acid sequence shown in SEQ ID NO:5. After the PCR, the amplifiedDNA was subjected to electrophoresis using 1% agarose gel, and a DNAfragment of about 4000 bp was purified using QIAquick Gel Extraction Kit(manufactured by QIAGEN).

The purified DNA fragment was ligated into a cloning vector pCR-Blunt(manufactured by Invitrogen). E. coli was transformed with the resultingligation product, and the plasmid was then recovered. The amplified genefragment was confirmed to have the same sequence as that of interest bysequencing. The plasmid having the same sequence as that of interest wastreated with restriction enzymes NotI and XhoI, and purified usingQIAquick Gel Extraction Kit, followed by inserting the gene sequence ofinterest into a mammalian expression vector PCDNA3.1 (manufactured byInvitrogen) that had been treated with restriction enzymes NotI andXhoI. Use of this vector enables production of the PDS5A protein inmammalian cells.

To 100 μg of the thus prepared plasmid DNA, 50 μg of gold particles(manufactured by Bio Rad), 100 μl spermidine (manufactured by SIGMA) and100 μl of 1 M CaCl₂ (manufactured by SIGMA) were added, and theresulting mixture was stirred by vortexing, followed by leaving themixture to stand for 10 minutes at room temperature (the resultingparticles are hereinafter referred to as gold-DNA particles). Themixture was then centrifuged at 3000 rpm for 1 minute and thesupernatant was discarded, followed by rinsing the precipitate 3 timeswith 100% ethanol (manufactured by WAKO). To the gold-DNA particles, 6ml of 100% ethanol was added, and the resulting mixture was sufficientlystirred by vortexing, followed by pouring the gold-DNA particles intoTefzel Tubing (manufactured by Bio Rad) and allowing the particles toprecipitate on the wall surface. The ethanol in the Tefzel Tubing towhich the gold-DNA particles are attached was dried in the air, and thetube was cut into pieces having a length appropriate for a gene gun.

(2) Anti-Tumor Effect of PDS5A by DNA Vaccine Method

Each of a murine neuroblastoma cell line N2a and a colon cancer cellline CT26 were subcutaneously transplanted to 10 individuals of A/J mice(7 weeks old, male, purchased from Japan SLC) and Balb/c mice (7 weeksold, male, purchased from Japan SLC) in an amount of 1×10⁶ cells. Theabove prepared tube was fixed in a gene gun, and a pressure of 400 psiwas applied using pure helium gas to perform percutaneous administrationof the DNA vaccine to the abdominal cavity of each mouse whose hair hadbeen shaved, which administration was repeated a total of 3 times atintervals of 7 days (this corresponds to 2 μg/individual in terms of thedose of the inoculated amount of the plasmid DNA) to evaluate theanti-tumor effect (therapeutic model). Further, in a similar manner, theDNA vaccine was subcutaneously administered to each of 10 individuals ofA/J mice and Balb/c mice a total of 3 times at intervals of 7 days, andN2a cells or CT26 cells were then transplanted to each mouse to evaluatethe anti-tumor effect (prophylactic model). As a control, a plasmid DNAto which the PDS5A gene was not inserted was administered to 10individuals in each model.

The anti-tumor effect was evaluated based on the size of the tumor(major axis×minor axis²/2) and the ratio of living mice. The results areshown in FIGS. 4 to 11. As a result of this study, in the therapeuticmodel using the neuroblastoma cell line, the size of the tumor on Day 41was 569 mm³ and 109 mm³ in the control group and the PDS5Aplasmid-administered group, respectively, indicating significantreduction of the tumor in the PDS5A plasmid-administered group (FIG. 4).Similarly, in the prophylactic model using the neuroblastoma cell line,the size of the tumor on Day 43 was 476 mm³ and 0 mm³ in the controlgroup and the PDS5A plasmid-administered group, respectively, indicatingcomplete regression of the tumor in the PDS5A plasmid-administered group(FIG. 5). Further, in the therapeutic model using the colon cancer cellline, the size of the tumor on Day 41 was 589 mm³ and 189 mm³ in thecontrol group and the PDS5A plasmid-administered group, respectively,indicating significant reduction of the tumor in the PDS5Aplasmid-administered group (FIG. 8). Further, in the prophylactic modelusing the colon cancer cell line, the size of the tumor on Day 43 was397 mm³ and 43 mm³ in the control group and the PDS5Aplasmid-administered group, respectively, indicating significantreduction of the tumor in the PDS5A plasmid-administered group (FIG. 9).Based on observation of the process of survival in the both models usingthe neuroblastoma cell line, while all the cases in the control groupdied by Day 84 after the administration, 60% of the mice were alive atthat time in the PDS5A plasmid-administered group (FIG. 6). Further, inthe prophylactic model, while all the cases in the control group died byDay 90 after the administration, all the mice were alive at that time inthe PDS5A plasmid-administered group (FIG. 7). Further, based onobservation of the process of survival in the both models using thecolon cancer cell line, while all the cases in the control group died byDay 84 after the administration, 40% of the mice were alive at that timein the PDS5A plasmid-administered group (FIG. 10). Further, in theprophylactic model, while all the cases in the control group died by Day90 after the administration, 80% of the mice were alive at that time inthe PDS5A plasmid-administered group (FIG. 11).

In the above results, a significantly higher anti-tumor effect wasobserved in the PDS5A-plasmid administered group than in the controlgroup, and, by this observation, it was revealed that PDS5A is a cancerantigen having a strong cancer antigenicity and effective for therapyand prophylaxis of cancer.

Example 3 Induction of Peptide Epitope-Reactive CD8-Positive T Cells

For prediction of an HLA-A0201-binding motif in the amino acid sequenceof the human PDS5A protein, a computer-based prediction program usingthe known BIMAS software (available athttp://bimas.dcrt.nih.gov/molbio/hla_bind/) was used to analyze theamino acid sequences shown in SEQ ID NOs:4 and 44, and thereby thepolypeptides shown in SEQ ID NOs:27 to 35, which were expected to becapable of binding to the HLA class I molecule, were selected.

From an HLA-A0201-positive healthy individual, peripheral blood wasseparated, and the peripheral blood was overlaid on Lymphocyteseparation medium (OrganonpTeknika, Durham, N.C.), followed bycentrifuging the resultant at 1,500 rpm at room temperature for 20minutes. A fraction containing peripheral blood mononuclear cells(PBMCs) was recovered and washed 3 times in a cold phosphate buffer, toobtain PBMCs. The obtained PBMCs were suspended in 20 ml of AIM-V medium(Life Technologies, Inc., Grand Island, N.Y., USA), and the cells wereallowed to attach to a culture flask (Falcon) at 37° C. under 5% CO₂ for2 hours. Unattached cells were used for preparation of T cells, andattached cells were used for preparation of dendritic cells.

The attached cells were cultured in AIM-V medium in the presence of IL-4(1000 U/ml) and GM-CSF (1000 U/ml). The medium was replaced 6 days laterwith AIM-V medium supplemented with IL-4 (1000 U/ml), GM-CSF (1000U/ml), IL-6 (1000 U/ml, Genzyme, Cambridge, Mass.), IL-1β (10 ng/ml,Genzyme, Cambridge, Mass.) and TNF-α (10 ng/ml, Genzyme, Cambridge,Mass.), and the culture was carried out for additional 2 days to obtaina population of unattached cells, which were employed as the dendriticcells.

The prepared dendritic cells were suspended in AIM-V medium at a celldensity of 1×10⁶ cells/ml, and each of the selected polypeptides wasadded at a concentration of 10 μg/ml to the suspension. Using a 96-wellplate, the cells were cultured at 37° C. under 5% CO₂ for 4 hours. Afterthe culture, X-ray irradiation (3000 rad) was carried out, and the cellswere washed with AIM-V medium, followed by being suspended in AIM-Vmedium supplemented with 10% human AB serum (Nabi, Miami, Fla.), IL-6(1000 U/ml) and IL-12 (10 ng/ml, Genzyme, Cambridge, Mass.). The cellswere placed in a 24-well plate in an amount of 1×10⁵ cells/well.Further, the prepared T cell population was added to each well in anamount of 1×10⁶ cells, and cultured at 37° C. under 5% CO₂. Each culturesupernatant was discarded 7 days later, and dendritic cells obtained inthe same manner as described above by treatment with each polypeptideand the subsequent X-ray irradiation were suspended in AIM-V mediumsupplemented with 10% human AB serum (Nabi, Miami, Fla.), IL-7 (10 U/ml,Genzyme, Cambridge, Mass.) and IL-2 (10 U/ml, Genzyme, Cambridge, Mass.)(cell density, 1×10⁵ cells/ml), which suspension was then added to the24-well plate in an amount of 1×10⁵ cells/well, followed by furtherculturing the cells. The same operation was repeated 4 to 6 times atintervals of 7 days, and stimulated T cells were then recovered,followed by confirmation of induction of CD8-positive T cells by flowcytometry.

Example 4 Determination of Cytotoxic T Cell Antigen Epitope in PDS5Athat Stimulates HLA-A0201-Positive CD8-Positive T Cells

Among the induced T cells in the respective wells, growth of T cellsstimulated by each of the polypeptides of SEQ ID NOs:27 to 35 wasconfirmed by counting of the cell number under the microscope. In orderto investigate the specificity of the respective T cells, whose growthwas confirmed, to each polypeptide used for pulsing, 5×10³ T cells wereadded with respect to 5×10⁴ T2 cells expressing the HLA-A0201 molecule(Salter R D et al., Immunogenetics, 21: 235-246 (1985), purchased fromATCC) pulsed with the polypeptide (each polypeptide was added to AIM-Vmedium at a concentration of 10 μg/ml, and the cells were culturedtherein at 37° C. under 5% CO₂ for 4 hours), and the cells were culturedin AIM-V medium supplemented with 10% human AB serum in a 96-well platefor 24 hours. After recovering the supernatant after the culture, theamount of production of IFN-γ was measured by the ELISA method. As aresult, higher production of IFN-γ was confirmed in the culturesupernatants in the wells containing T2 cells pulsed with the respectivepolypeptides shown in SEQ ID NOs:27 to 35 compared to the culturesupernatants in the wells containing T2 cells which were not pulsed witha polypeptide (FIG. 12). Thus, it was revealed that each of thepolypeptides of SEQ ID NOs:27 to 35 is a T cell epitope peptide having acapacity to stimulate and proliferate HLA-A0201-positive CD8-positive Tcells, to induce production of IFN-γ. On the other hand, in the casewhere the polypeptide having the amino acid sequence shown in SEQ IDNO:36, which is outside the scope of the present invention, was added toperform the above-described treatment, no production of IFN-γ could beconfirmed (FIG. 12).

Subsequently, whether or not the respective polypeptides shown in SEQ IDNOs:27 to 35, which are polypeptides to be used in the presentinvention, are presented on HLA-A0201 molecules on HLA-A0201-positivetumor cells expressing PDS5A, and whether or not CD8-positive cellsstimulated with the polypeptides can damage HLA-A0201-positive tumorcells expressing PDS5A, were studied.

In a 50-ml centrifuge tube, 10⁵ cells of a malignant brain tumor cellline T98G, whose expression of PDS5A had been confirmed (Stein G H etal., J. Cell Physiol., 99: 43-54 (1979), purchased from ATCC), werecollected, and 100 μCi of chromium 51 was added to the tube, followed byincubation at 37° C. for 2 hours. Subsequently, the cells were washed 3times with AIM-V medium supplemented with 10% human AB serum, and placedin a 96-well V-bottomed plate in an amount of 10³ cells per well,followed by further addition, to each well, of 10⁵, 5×10⁴, 2.5×10⁴ or1.25×10⁴ HLA-A0201-positive CD8-positive T cells suspended in AIM-Vmedium supplemented with 10% human AB serum, which cells were stimulatedwith the respective polypeptides shown in SEQ ID NOs:27 to 35. The cellswere then cultured at 37° C. under 5% CO₂ for 4 hours. Thereafter, theamount of chromium 51 released from damaged tumor cells into the culturesupernatant was measured, and thereby the cytotoxic activity of theCD8-positive T cells stimulated with each of the polypeptides shown inSEQ ID NOs:27 to 35 was calculated.

As a result, it was revealed that the HLA-A0201-positive CD8-positive Tcells stimulated with the respective polypeptides shown in SEQ ID NOs:27to 35 have the cytotoxic activity against T98G (FIG. 13). Therefore, itbecame clear that the polypeptides shown in SEQ ID NOs:27 to 35, whichare polypeptide to be used in the present invention, are presented onHLA-A0201 molecules on HLA-A0201-positive tumor cells expressing PDS5A,and that these polypeptides have a capacity to induce CD8-positivecytotoxic T cells which can damage such tumor cells. On the other hand,in the case where the polypeptide having the amino acid sequence shownin SEQ ID NO:36, which is outside the scope of the present invention,was added to perform the above-described treatment, no cytotoxicactivity could be observed (FIG. 13).

The cytotoxic activity was determined by, as described above, mixing 10⁵CD8-positive T cells stimulated and induced with each of the peptides ofthe present invention and 10³ cells of a malignant brain tumor cell lineT98G to which chromium 51 was incorporated; culturing the resultant for4 hours; measuring the amount of chromium 51 released into the culturemedium after the culturing; and calculating the cytotoxic activity ofthe CD8-positive T cells against T98G according to the followingequation*.

cytotoxic activity (%)=(Amount of chromium 51 released from T98G whenCD8-positive T cells were added)/(Amount of chromium 51 released fromthe target cells to which 1N hydrochloric acid wasadded)×100.  *Equation:

Example 5 Preparation, and Evaluation of Pharmacological Effect, ofRecombinant PDS5A Protein; Detection of Cancer; and Cancer Diagnosis (1)Preparation of Recombinant PDS5A Protein

Based on the gene of SEQ ID NO:1 obtained in Example 1, a recombinantprotein was prepared by the following method. Regents and an attachedbuffer were mixed such that 1 μl of the vector obtained in Example 1which was prepared from the phagemid solution and subjected to thesequence analysis, 0.4 μM each of two kinds of primers having the NotIand XhoI restriction sites (shown in SEQ ID NOs:37 and 38), 0.2 mM dNTPand 1.25 U PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co.,Ltd.) were contained in a total volume of 50 and PCR was carried out byrepeating 30 times the cycle of 98° C. for 10 seconds, 55° C. for 15seconds and 72° C. for 4 minute using a Thermal Cycler (manufactured byBIO RAD). The above-described two kinds of primers were those foramplification of the region encoding the full-length of the amino acidsequence shown in SEQ ID NO:2. After the PCR, the amplified DNA wassubjected to electrophoresis using 1% agarose gel, and a DNA fragment ofabout 4000 bp was purified using QIAquick Gel Extraction Kit(manufactured by QIAGEN).

The purified DNA fragment was ligated into a cloning vector pCR-Blunt(manufactured by Invitrogen). E. coli was transformed with the resultingligation product, and the plasmid was then recovered. The amplified genefragment was confirmed to have the same sequence as that of interest bysequencing. The plasmid having the same sequence as that of interest wastreated with restriction enzymes NotI and XhoI, and purified usingQIAquick Gel Extraction Kit, followed by inserting the gene sequence ofinterest into an expression vector for E. coli, pET30a (manufactured byNovagen) that had been treated with restriction enzymes NotI and XhoI.Use of this vector enables production of a His tag-fused recombinantprotein. E. coli for expression, BL21 (DE3), was transformed with thisplasmid, and expression was induced with 1 mM IPTG, to allow expressionof the protein of interest in E. coli.

Further, based on the gene of SEQ ID NO:43, a recombinant protein ofhuman PDS5A was prepared by the following method. Regents and anattached buffer were mixed such that 1 μl of the cDNA prepared inExample 1 whose expression could be confirmed with cDNAs from varioustissues and cells by the RT-PCR method, 0.4 μl each of two kinds ofprimers having the NotI and XhoI restriction sites (shown in SEQ IDNOs:39 and 40), 0.2 mM dNTP and 1.25 U PrimeSTAR HS polymerase(manufactured by Takara Shuzo Co., Ltd.) were contained in a totalvolume of 50 μl, and PCR was carried out by repeating 30 times the cycleof 98° C. for 10 seconds, 55° C. for 15 seconds and 72° C. for 4 minuteusing a Thermal Cycler (manufactured by BIO RAD). The above-describedtwo kinds of primers were those for amplification of the region encodingthe full-length of the amino acid sequence shown in SEQ ID NO:44. Afterthe PCR, the amplified DNA was subjected to electrophoresis using 1%agarose gel, and a DNA fragment of about 4000 bp was purified usingQIAquick Gel Extraction Kit (manufactured by QIAGEN).

The purified DNA fragment was ligated into a cloning vector pCR-Blunt(manufactured by Invitrogen). E. coli was transformed with the resultingligation product, and the plasmid was then recovered. The amplified genefragment was confirmed to have the same sequence as that of interest bysequencing. The plasmid having the same sequence as that of interest wastreated with restriction enzymes NotI and XhoI, and purified usingQIAquick Gel Extraction Kit, followed by inserting the gene sequence ofinterest into an expression vector for E. coli, pET30a (manufactured byNovagen) that had been treated with restriction enzymes NotI and XhoI.Use of this vector enables production of a His tag-fused recombinantprotein. E. coli for expression, BL21 (DE3), was transformed with thisplasmid, and expression was induced with 1 mM IPTG, to allow expressionof the protein of interest in E. coli.

Further, based on the gene of SEQ ID NO:5, a recombinant protein ofmurine PDS5A was prepared by the following method. Regents and anattached buffer were mixed such that 1 μl of the cDNA prepared inExample 1 whose expression could be confirmed with cDNAs from varioustissues and cells by the RT-PCR method, 0.4 μM each of two kinds ofprimers having the NotI and XhoI restriction sites (shown in SEQ IDNOs:41 and 42), 0.2 mM dNTP and 1.25 U PrimeSTAR HS polymerase(manufactured by Takara Shuzo Co., Ltd.) were contained in a totalvolume of 50 μl, and PCR was carried out by repeating 30 times the cycleof 98° C. for 10 seconds, 55° C. for 15 seconds and 72° C. for 4 minuteusing a Thermal Cycler (manufactured by BIO RAD). The above-describedtwo kinds of primers were those for amplification of the region encodingthe full-length of the amino acid sequence shown in SEQ ID NO:6. Afterthe PCR, the amplified DNA was subjected to electrophoresis using 1%agarose gel, and a DNA fragment of about 4000 bp was purified usingQIAquick Gel Extraction Kit (manufactured by QIAGEN).

The purified DNA fragment was ligated into a cloning vector pCR-Blunt(manufactured by Invitrogen). E. coli was transformed with the resultingligation product, the plasmid was then recovered. The amplified genefragment was confirmed to have the same sequence as that of interest bysequencing. The plasmid having the same sequence as that of interest wastreated with restriction enzymes NotI and XhoI, and purified usingQIAquick Gel Extraction Kit, followed by inserting the gene sequence ofinterest into an expression vector for E. coli, pET30a (manufactured byNovagen) that had been treated with restriction enzymes NotI and XhoI.Use of this vector enables production of a His tag-fused recombinantprotein. E. coli for expression, BL21 (DE3), was transformed with thisplasmid, and expression was induced with 1 mM IPTG, to allow expressionof the protein of interest in E. coli.

(2) Purification of PDS5A Protein

Each of the above obtained recombinant E. coli that expresses SEQ IDNO:2, SEQ ID NO:44 or SEQ ID NO:6 was cultured in LB medium supplementedwith 100 μg/ml ampicillin at 37° C. until the absorbance at 600 nmreached about 0.7, and then isopropyl-β-D-1-thiogalactopyranoside wasadded thereto to a final concentration of 1 mM, followed by furtherculturing the recombinant E. coli at 37° C. for 4 hours. Subsequently,the bacterial cells were collected by centrifugation at 4,800 rpm for 10minutes. The pellet of the cells was suspended in phosphate-bufferedsaline and further subjected to centrifugation at 4,800 rpm for 10minutes to wash the bacterial cells.

The bacterial cells were suspended in 50 mM Tris-HCl buffer (pH 8.0) andsubjected to sonication on ice. The liquid obtained by the sonication ofE. coli was centrifuged at 6000 rpm for 20 minutes, to obtain thesupernatant as the soluble fraction and the precipitate as the insolublefraction.

The insoluble fraction was suspended in 50 mM Tris-HCl buffer (pH 8.0)and centrifuged at 6000 rpm for 15 minutes. This operation was repeatedtwice to perform an operation of removal of proteases.

The residue was suspended in 50 mM Tris-HCl buffer (pH 8.0) supplementedwith 6 M guanidine hydrochloride and 0.15 M sodium chloride, and left tostand at 4° C. for 20 hours to denature proteins. Thereafter, thesuspension was centrifuged at 6000 rpm for 30 minutes, and the obtainedsoluble fraction was placed in a nickel chelate column prepared by aconventional method (carrier: Chelating Sepharose (trademark) Fast Flow(GE Health Care); column volume: 5 mL; equilibration buffer: 50 mMTris-HCl buffer (pH 8.0) supplemented with 6M guanidine hydrochlorideand 0.15 M sodium chloride), followed by leaving the resultant to standat 4° C. overnight to allow adsorption of the proteins to thenickel-chelated carrier. This column carrier was centrifuged at 1500 rpmfor 5 minutes and the supernatant was then recovered. The column carrierwas suspended in phosphate-buffered saline and refilled into the column.

The fraction not adsorbed to the column was washed with 10 columnvolumes of 0.1 M acetate buffer (pH 4.0) supplemented with 0.5 M sodiumchloride, and immediately thereafter, proteins were eluted with 0.1 Macetate buffer (pH 3.0) supplemented with 0.5 M sodium chloride, toobtain a purified fraction, which was used later as a material for anadministration test. The protein of interest in each eluted fraction wasconfirmed by Coomassie staining carried out according to a conventionalmethod.

The buffer of the purified preparation obtained by the above method wasreplaced with a reaction buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM CaCl₂(pH8.0)), and the resulting sample was subjected to cleavage of the Histag with factor Xa protease and purification of the protein of interest,using Factor Xa Cleavage Capture Kit (manufactured by Novagen) inaccordance with the protocol attached to the kit. Subsequently, thebuffer of 12 ml of the purified preparation obtained by the above methodwas replaced with a physiological phosphate buffer (manufactured byNissui Pharmaceutical) using ultrafiltration NANOSEP 10K OMEGA(manufactured by PALL), and the resulting sample was subjected toaseptic filtration through HT Tuffryn Acrodisc 0.22 μm (manufactured byPALL) and used in the experiment.

(3) Anti-Tumor Effect of Recombinant Murine PDS5A Protein inTumor-Bearing Mouse

The murine neuroblastoma cell line N2a was subcutaneously transplantedto A/J mice (7 weeks old, male, purchased from Japan SLC) in an amountof 1×10⁶ cells. When the tumor volume reached an average of 50 to 100mm³ (typically 7 days after the inoculation of the tumor), the mice wererandomly divided into groups each of which contains 10 individuals, andsubjected to evaluation of the anti-tumor effect of the recombinantmurine PDS5A protein (therapeutic model). With 100 μg (0.5 ml) of therecombinant murine PDS5A protein purified as described above, 50 μg ofpoly I:C was mixed to prepare a therapeutic agent for cancer, and thistherapeutic agent was subcutaneously administered to the tumor-bearingmice a total of 3 times at intervals of 1 week. As a result, on Day 31after the administration of the therapeutic agent for cancer, completeregression of the tumor was achieved. On the other hand, in the negativecontrol group to which PBS(−) was administered and the group to whichpoly I:C alone (50 μg) was administered, the mean tumor volumes on Day31 after the administration were 1657 mm³ and 932 mm³, respectively.

Further, a therapeutic agent for cancer wherein 100 μg (0.5 ml) of therecombinant murine PDS5A protein and 50 μg poly I:C were mixed wasprepared, and subcutaneously administered to A/J mice a total of 3 timesat intervals of 1 week, followed by transplantation of 1×10⁶ N2a cellsto the mice and evaluation of the anti-tumor effect (prophylacticmodel). Ten individuals were included in each group, and, as controlsfor comparison, a negative control group to which PBS(−) wasadministered and a group to which poly I:C alone (50 μg) wasadministered were provided. As a result, in the group to which thetherapeutic agent for cancer was administered, no development of a tumorwas observed even on Day 40 after the administration of the therapeuticagent for cancer. On the other hand, in the negative control group towhich PBS(−) was administered and the group to which poly I:C alone (50μg) was administered, the mean tumor volumes on Day 40 after theadministration were 1989 mm³ and 1843 mm³, respectively.

The same experiment was carried out also for a colon cancer model. Thecolon cancer cell line CT26 was subcutaneously transplanted to Balb/cmice (7 weeks old, male, purchased from Japan SLC) in an amount of 1×10⁶cells. When the tumor volume reached an average of 50 to 100 mm³(typically 7 days after the inoculation of the tumor), the mice wererandomly divided into groups each of which contains 10 individuals, andsubjected to evaluation of the anti-tumor effect of the recombinantmurine PDS5A protein (therapeutic model). With 100 μg (0.5 ml) of therecombinant murine PDS5A protein purified as described above, 50 μg ofpoly I:C was mixed to prepare a therapeutic agent for cancer, and thistherapeutic agent was subcutaneously administered to the tumor-bearingmice a total of 3 times at intervals of 1 week. As a result, on Day 24after the administration of the therapeutic agent for cancer, completeregression of the tumor was achieved. On the other hand, in the negativecontrol group to which PBS(−) was administered and the group to whichpoly I:C alone (50 μg) was administered, the mean tumor volumes on Day24 after the administration were 1449 mm³ and 835 mm³, respectively.

Further, a therapeutic agent for cancer wherein 100 μg (0.5 ml) of therecombinant murine PDS5A protein and 50 μg poly I:C were mixed wasprepared, and subcutaneously administered to Balb/c mice a total of 3times at intervals of 1 week, followed by transplantation of 1×10⁶ CT26cells to the mice and evaluation of the anti-tumor effect (prophylacticmodel). Ten individuals were included in each group, and, as controlsfor comparison, a negative control group to which PBS(−) wasadministered and a group to which poly I:C alone (50 μg) wasadministered were provided. As a result, in the group to which thetherapeutic agent for cancer was administered, no development of a tumorwas observed even on Day 31 after the administration of the therapeuticagent for cancer. On the other hand, in the negative control group towhich PBS(−) was administered and the group to which poly I:C alone (50μg) was administered, the mean tumor volumes on Day 31 after theadministration were 1781 mm³ and 1675 mm³, respectively.

From these results, it was revealed that the recombinant PDS5A proteinis effective for therapy and prophylaxis of cancer.

(4) Anti-Tumor Effect of Recombinant PDS5A Protein in Tumor-Bearing Dog

The anti-tumor effect of the recombinant protein described in Example 5below in 3 individuals of tumor-bearing patient dogs (3 individualshaving a mammary gland tumor) having a tumor mass in the epidermis wasevaluated. Before administration, the antibody titer against therecombinant protein in the serum of each patient dog was measured by themethod described in Example 5 (3), and, as a result, an antibody titerhigher than that of a healthy dog was detected. From these results, itwas suggested that the protein having the amino acid sequence shown inSEQ ID NO:2 was expressed as a cancer antigen in the tumor tissue in theliving body of these tumor-bearing patient dogs.

With 500 μg (2.5 ml) each of the recombinant PDS5A proteins (dog-derivedand human-derived) purified as described above, the same amount ofFreund's incomplete adjuvant (manufactured by Wako Pure ChemicalIndustries, Ltd.) was mixed to prepare 2 kinds of therapeutic agents forcancer, each of which was administered to a regional lymph node in thevicinity of the tumor a total of 3 times at 1-week intervals. As aresult, complete regression of the tumor, which had had a size of about500 mm³ or 1000 mm³ at the time of administration of each therapeuticagent for cancer, was achieved on Day 13 or Day 21, respectively. On theother hand, in the negative control group to which PBS(−) wasadministered, the tumor volume, which had been about 800 mm³ at the timeof administration of PBS, became 1625 mm³ on Day 21 after theadministration.

With 500 μg (2.5 ml) of the canine recombinant PDS5A protein purified asdescribed in Example 5 below, the same amount of Freund's incompleteadjuvant (manufactured by Wako Pure Chemical Industries, Ltd.) was mixedto prepare a therapeutic agent for cancer, and this therapeutic agentwas subcutaneously administered in the vicinity of the tumor in 1individual each of patient dogs suffering from perianal adenocarcinomaand epidermal squamous cell carcinoma a total of 4 times at 1-weekinterval. As a result, complete regression of the tumor, which had had asize of about 370 mm³ or 280 mm³, respectively, at the time ofadministration of the therapeutic agent for cancer, was achieved on Day35 or Day 42, respectively.

(5) Detection of Cancer Using Recombinant PDS5A Protein

Blood was collected from 112 patient dogs wherein malignant tumor wasfound and 30 healthy dogs, and sera were separated therefrom. Using thecanine PDS5A protein (SEQ ID NO:2) prepared in the above-described (2),the titer of antibodies specifically reactive with the protein in eachserum was measured by the ELISA method. Immobilization of the preparedprotein was carried out by placing 100 μL/well of the recombinantprotein solution diluted to 5 μg/mL with phosphate-buffered saline in a96-well Immobilizer Amino plate (manufactured by Nunc), followed byleaving the plate to stand at 4° C. overnight. Blocking was carried outby adding 100 μL of 50 mM sodium bicarbonate buffer (pH 8.4)supplemented with 3% BSA (bovine serum albumin, manufactured bySigma-Aldrich Co.) (hereinafter referred to as the blocking solution) toeach well and shaking the plate at room temperature for 1 hour. The serawere 1000-fold diluted with the blocking solution and added to the wellsin an amount of 100 μL/well, and the plate was shaken at roomtemperature for 3 hours to allow the reaction to proceed. The wells werewashed 3 times with phosphate-buffered saline supplemented with 0.05%Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.)(hereinafter referred to as PBS-T), and 100 μL/well of an HRP-modifiedanti-dog IgG antibody (Goat anti Dog IgG-h+I HRP conjugated:manufactured by BETHYL Laboratories) 3000-fold diluted with the blockingsolution was added thereto, followed by shaking the plate at roomtemperature for 1 hour to allow the reaction to proceed. After washingthe wells 3 times with PBS-T, 100 μl/well of an HRP substrate TMB(1-Step Turbo TMB (tetramethylbenzidine), PIERCE) was added, and theenzyme-substrate reaction was allowed to proceed at room temperature for30 minutes. Thereafter, 100 μl/well of 0.5 M sulfuric acid solution(manufactured by Sigma-Aldrich Japan) was added to the wells to stop thereaction, and the absorbance at 450 nm was measured using a microplatereader. To prepare controls for comparison, experiments were carried outin the same manner as described above except that the preparedrecombinant protein was not immobilized or except that the tumor-bearingdog serum was not reacted.

All the 112 samples used for the above-described cancer diagnosis werethose which had been definitely diagnosed as malignant by pathologicaldiagnosis using extirpated tumor tissues.

Specifically, the samples were those diagnosed as cancers such asmalignant melanoma, malignant mixed tumor, hepatocellular carcinoma,basal cell carcinoma, intraoral tumor, perianal adenocarcinoma, anal sactumor, anal sac apocrine carcinoma, Sertoli cell tumor, vulva cancer,sebaceous adenocarcinoma, sebaceous epithelioma, sebaceous adenoma,sweat gland carcinoma, intranasal adenocarcinoma, nasal adenocarcinoma,thyroid cancer, colon cancer, bronchial adenocarcinoma, adenocarcinoma,ductal carcinoma, mammary adenocarcinoma, combined mammaryadenocarcinoma, mammary gland malignant mixed tumor, intraductalpapillary adenocarcinoma, fibrosarcoma, hemangiopericytoma,osteosarcoma, chondrosarcoma, soft tissue sarcoma, histiocytic sarcoma,myxosarcoma, undifferentiated sarcoma, lung cancer, mastocytoma,cutaneous leiomyoma, intra-abdominal leiomyoma, leiomyoma, squamous cellcarcinoma, chronic lymphocytic leukemia, lymphoma, gastrointestinallymphoma, digestive organ lymphoma, small cell or medium cell lymphoma,adrenomedullary tumor, granulosa cell tumor and pheochromocytoma.

Sera from these cancer-bearing dogs showed significantly higher antibodytiters against the recombinant protein than sera from the healthy dogs.It was revealed that, by diagnosing a sample showing a value not lessthan twice as high as the average value in healthy dogs as malignant, 94samples, which corresponds to 83.9% of the malignant cases, could besuccessfully diagnosed as malignant. The types of the cancers in these94 samples were as described below. It should be noted that, although apart of the samples were suffering from a plurality of types of cancers,each value shown below is the cumulative total for each type of cancer.

Malignant melanoma, 5 cases; lymphoma, 10 cases; granulosa cell tumor, 1case; hepatocellular carcinoma, 3 cases; malignant testicular tumor, 3cases; intraoral tumor, 3 cases; perianal adenocarcinoma, 5 cases;sarcoma, 9 cases; mammary adenocarcinoma, 35 cases; lung cancer, 1 case;ductal carcinoma, 4 cases; sebaceous adenocarcinoma, 2 cases;mastocytoma, 5 cases; leiomyosarcoma, 1 case; squamous cell carcinoma, 4cases; malignant mixed tumor, 2 cases; and hemangiopericytoma, 1 case.

When cancer diagnosis was carried out in the same manner as describedabove using the human PDS5A protein (SEQ ID NO:44) prepared in theabove-described (2), a similar result was obtained.

From the above results, it was revealed that, by using the PDS5A proteinto measure the titer of antibodies specifically reactive with theprotein in the serum, detection and diagnosis of cancer is possible.

INDUSTRIAL APPLICABILITY

The immunity-inducing agent of the present invention comprising apolypeptide that exerts an anti-tumor activity against various types ofcancers is useful for therapy and/or prophylaxis of cancer, and/ordetection of cancer.

1. An immunity-inducing agent comprising as an effective ingredient(s)at least one polypeptide selected from the polypeptides (a) to (c)below, said polypeptide(s) having an immunity-inducingactivity/activities, or as an effective ingredient(s) a recombinantvector(s) which comprise(s) a polynucleotide(s) encoding saidpolypeptide(s) and is/are capable of expressing said polypeptide(s) invivo: (a) a polypeptide consisting essentially of not less than 7consecutive amino acids in any one of the amino acid sequences shown inSEQ ID NOs:2, 4, 6, 8, 10, 12 and 44; (b) a polypeptide having asequence identity of not less than 90% with said polypeptide (a) andconsisting essentially of not less than 7 amino acids; and (c) apolypeptide comprising said polypeptide (a) or (b) as a partial sequencethereof.
 2. The immunity-inducing agent according to claim 1, whereinsaid polypeptide (b) has a sequence identity of not less than 95% withsaid polypeptide (a).
 3. The immunity-inducing agent according to claim1, wherein each of said polypeptide(s) having an immunity-inducingactivity/activities is a polypeptide consisting essentially of not lessthan 7 consecutive amino acids in any one of the amino acid sequencesshown in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 44, or a polypeptidecomprising said polypeptide as a partial sequence thereof or apolypeptide having the same amino acid sequence as a polypeptideconsisting essentially of not less than 7 consecutive amino acids in anyone of the amino acid sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12and 44 except that one or several amino acids are deleted, substitutedand/or added, or a polypeptide comprising said polypeptide as a partialsequence thereof.
 4. The immunity-inducing agent according to claim 3,wherein each of said polypeptide(s) having an immunity-inducingactivity/activities is a polypeptide having any one of the amino acidsequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12 and
 44. 5. Theimmunity-inducing agent according to claim 3, wherein each of saidpolypeptide(s) having an immunity-inducing activity/activities is apolypeptide consisting essentially of not less than 7 consecutive aminoacids in the region of aa111-140, aa211-240, aa248-278, aa327-357,aa459-522, aa909-972, aa959-1022, aa994-1057 or aa1018-1080 in any oneof the amino acid sequences shown in SEQ ID NOs:2, 6, 8, 10, 12 and 44,or a polypeptide comprising said polypeptide as a partial sequencethereof or a polypeptide having the same amino acid sequence as apolypeptide consisting essentially of not less than 7 consecutive aminoacids in the region of aa111-140, aa211-240, aa248-278, aa327-357,aa459-522, aa909-972, aa959-1022, aa994-1057 or aa1018-1080 in any oneof the amino acid sequences shown in SEQ ID NOs:2, 6, 8, 10, 12 and 44except that one or several amino acids are deleted, substituted and/oradded, or a polypeptide comprising said polypeptide as a partialsequence thereof.
 6. The immunity-inducing agent according to claim 5,wherein each of said polypeptide(s) having an immunity-inducingactivity/activities is a polypeptide having any one of the amino acidsequences shown in SEQ ID NOs:27 to 35, or a polypeptide comprising saidpolypeptide as a partial sequence thereof and having 10 to 12 amino acidresidues; or a polypeptide having the same amino acid sequence as apolypeptide having any one of the amino acid sequences shown in SEQ IDNOs:27 to 35 except that one or several amino acids are deleted,substituted and/or added, or a polypeptide comprising said polypeptideas a partial sequence thereof and having 10 to 12 amino acid residues.7. The immunity-inducing agent according to claim 1, for prophylaxis ofa cancer in an animal.
 8. The immunity-inducing agent according to claim5, for therapy of a cancer in an animal.
 9. The immunity-inducing agentaccording to claim 7, wherein said cancer is a cancer expressing PDS5A.10. The immunity-inducing agent according to claim 7, wherein saidcancer is breast cancer, brain tumor, esophagus cancer, lung cancer,renal cancer, colon cancer, perianal adenocarcinoma, neuroblastoma orleukemia.
 11. The immunity-inducing agent according to claim 1, furthercomprising an immunoenhancer.
 12. An isolated antigen-presenting cellcomprising a complex between said polypeptide having animmunity-inducing activity according to claim 1 and an MHC molecule. 13.An isolated T cell which selectively binds to a complex between saidpolypeptide having an immunity-inducing activity according to claim 1and an MHC molecule.
 14. A polypeptide having any one of the amino acidsequences shown in SEQ ID NOs:27 to 35, or a polypeptide comprising saidpolypeptide as a partial sequence thereof and having 10 to 12 amino acidresidues; or a polypeptide having the same amino acid sequence as apolypeptide having any one of the amino acid sequences shown in SEQ IDNOs:27 to 35 except that one or several amino acids are deleted,substituted and/or added, or a polypeptide comprising said polypeptideas a partial sequence thereof and having 10 to 12 amino acid residues,which polypeptide has an immunity-inducing activity.
 15. A method fordetecting a cancer, said method comprising: measurement of expression ofa polypeptide having any one of the amino acid sequences shown in SEQ IDNOs:2, 4, 6, 8, 10, 12 and 44 or a polypeptide having a sequenceidentity of not less than 90% with said polypeptide, in a sampleseparated from a living body.
 16. A method for inducing immunity forand/or prophylaxis of a cancer in an animal, said method comprising:administering to the animal a therapeutically effective amount of an theimmunity-inducing agent according to claim
 1. 17. A method for inducingCD8+ cytotoxic T cells in an individual, said method comprising:administering to an individual a therapeutically effective amount of anthe immunity-inducing agent according to claim 1.